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Secondary metabolites from selected British Columbian marine organisms Tischler, Mark 1987

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SECONDARY METABOLITES FROM SELECTED BRITISH COLUMBIAN MARINE ORGANISMS By MARK TISCHLER A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1987 © Mark T i s c h l e r , 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date /9^// J ? ^ /9f? DE-6(3/81) - i i -TABLE OF CONTENTS Page ABSTRACT i v LIST OF TABLES v LIST OF FIGURES v i LIST OF SCHEMES v i i i LIST OF APPENDICES x ACKNOWLEDGEMENTS x i ABBREVIATIONS 1 x i i A. INTRODUCTION TO THE BRYOZOANS 1 i ) Biology 1 i i ) Natural Products Chemistry 3 B. SYNTHESIS OF PHIDOLOPIN AND DESMETHYLPHIDOLOPIN . . . . 16 C. NITROPHENOLS FROM NORTHEAST PACIFIC BRYOZOANS 45 D. DISCUSSION 54 E. INTRODUCTION TO THE SPONGES 63 - i i i -F. NOVEL STEROIDS FROM THE SPONGE Anthroarcuata gracea . . . 75 1. Introduction 75 2. I s o l a t i o n and Structure E l u c i d a t i o n 76 3. A. A4-3,6-diketosteroids 77 B. A-Nor s t e r o l s 89 C. Diosphenols 107 D. Biogenesis of Sponge Metabolites 115 G. EXPERIMENTAL 120 H. APPENDICES 139 I. BIBLIOGRAPHY 140 i v -ABSTRACT The two purine a l k a l o i d s , phidolopin (36) and desmethylphidolopin (37), o r i g i n a l l y i s o l a t e d from Phidolopora p a c i f i c a . were synthesized i n order to produce s u f f i c i e n t quantities of the natural products f o r extended b i o l o g i c a l and pharmacological screening and to confirm the previous s t r u c t u r a l assignment of 37 which was based on s p e c t r a l data. Various combinations of phidolopin (36), desmethylphidolopin (37), 4-hydroxymethyl-2-nitrophenol (38) and 4-methoxymethyl-2-nitrophenol (39) were i s o l a t e d from four d i f f e r e n t species of bryozoans, Diaperoecia  c a l i f o r n i c a . Heteropora alaskensis. T r i c e l l a r i a ternata and Hippodiplosia i n s c u l p t a . A dietary o r i g i n i s suggested f o r these metabolites. The red sponge, Anthoarcuata graceae. y i e l d e d s i x novel steroids i n c l u d i n g the ,6-diketosteroids 116, 117, the A-nor steroids anthosterone A (118) and anthosterone B (119) as w e l l as two diosphenol containing s t e r o i d s , 120 and 121. The proposed structures were based on a combination of s p e c t r a l analysis, chemical interconversions, synthe-s i s , and sing l e c r y s t a l X-ray d i f f r a c t i o n a n a l ysis. - V -LIST OF TABLES Table Page 1 80 MHz ^H NMR data on protected nitrophenols 41 and 42 22 2 80 MHz XH NMR data comparison of 53 and 54 . . . 30 3 80 MHz ^ NMR data for phidolopin (36) and desmethylphidolopin (37) 40 4 1 3 C NMR data comparison for 36 and 37 44 5 ln NMR data f o r i s o l a t e d nitrophenols 36, 37 . . 48 6 1H NMR data f o r i s o l a t e d nitrophenols 38, 39 . . 50 7 lU NMR data for A 4-3,6-ketosteroids (CDCI3) . . . 83 8 1 3 C NMR data for A 4-3,6-ketosteroids (CDCI3) . . . 86 9 1H NMR data for anthosterone A (118) and anthosterone B (119) 94 10 1 3 C NMR data for anthosterone A (118) and anthosterone B (119) 95 11 •'-H NMR comparison between 119 and 123 101 12 -^H NMR data for diosphenols 120 and 121 . . . . 110 13 Nitrophenols from Northeast P a c i f i c Bryozoans . . 131 - v i -LIST OF FIGURES Figure Page 1 80 MHz -^H NMR of compound 41 20 2 80 MHz 1H NMR of compound 42 21 3 80 MHz *H NMR of MOM protected desmethyl-phidolopin 55 33 4 80 MHz 1H NMR of MOM protected desmethyl-phidolopin 56 34 5 80 MHz XH NMR of phidolopin 36 38 6 80 MHz lH NMR of desmethylphidolopin 37 . . . . 39 7 100 MHz 1 3 C NMR of phidolopin 36 42 8 75 MHz 1 3 C NMR of desmethylphidolopin 37 . . . . 43 9 80 MHz XH NMR for 4-hydroxymethyl-2-nitrophenol 38 51 10 80 MHz 1H NMR for 4-methoxymethyl-2-nitrophenol 39 52 11 400 MHz *H NMR of compound 116 80 12 400 MHz 1H NMR of compound 122 81 13 75 MHz 1 3 C NMR of compound 122 82 14 400 MHz % NMR of compound 117 84 15 75 MHz 1 3 C NMR of compound 117 85 16 400 MHz 1H NMR of anthosterone A 118 90 17 75 MHz 1 3 C NMR of anthosterone A 118 91 18 400 MHz 1H NMR of anthosterone B 119 92 19 75 MHz 1 3 C NMR of anthosterone B 119 93a 20 APT spectrum f o r anthosterone B 119 93b 21 270 MHz -^H NMR of anthosterone B-acetate 123 . . 100 - v i i -22 A l l y l i c methylene system 102 23 SINEPT experiment on anthosterone B 119 . . . . 105 24 2D Homonuclear COSY on anthosterone B 119 . . . . 106 25 Computer generated ORTEP drawing of anthosterone A 118 106 26 400 MHz XH NMR of compound E 120 108 27 2D Homonuclear COSY spectrum of compound F 121 . . 112 28 400 MHz 1H NMR of compound F 121 113 - v i i i -LIST OF SCHEMES Scheme Page 1 S y n t h e t i c p l a n f o r p h i d o l o p i n (36) and desmethylphidolopin (37) 17 2 P r o t e c t i o n of 4-methyl-2-nitrophenol 40 . . . . 18 3 I n t e r p r e t a t i o n of the MS fragmentation of 41 . . 23 4 I n t e r p r e t a t i o n of the MS fragmentation of 42 . . 23 5 A l k y l a t i o n of t h e o p h y l l i n e (45) w i t h 47 . . . . 25 6 A l k y l a t i o n of t h e o p h y l l i n e w i t h 49 26 7 Bromination of p r o t e c t e d n i t r o p h e n o l 41 . . . . 26 8 MS fragmentation of compound 51 27 9 Bromination of MOM p r o t e c t e d n i t r o p h e n o l 42 . . . 28 10 A l k y l a t i o n of t h e o p h y l l i n e (45) w i t h 51 . . . . 29 11 Bromination and a l k y l a t i o n 31 12 I n t e r p r e t a t i o n of the MS fragmentation of 55 . . 35 13 A l k y l a t i o n of t h e o p h y l l i n e (45) under v a r i e d c o n d i t i o n s 36 14 Deprotection of 53 w i t h c h l o r o t r i m e t h y l s i l a n e . . 37 15 Deprotection of 55 and 56 w i t h d i l u t e a c i d . . . 37 16 MS fragmentation of p h i d o l o p i n (36) 49 17 MS fragmentation of desmethylphidolopin (37) . . 49 18 MS fragmentation of n i t r o p h e n o l 38 53 19 MS fragmentation of n i t r o p h e n o l 39 53 20 B i o s y n t h e s i s of x e s t o s t e r o l (95) 71 - i x -21 Biosynthetic conversion of c h o l e s t e r o l into 3B-hydroxymethyl-A-nor-cholestane 73 22 Jones oxidation of c h o l e s t e r o l (83) 79 23 MS fragmentation of compound B 117 87 24 MS fragmentation of anthosterone A (118) . . . . 97 25 MS fragmentation of anthosterone B (119) . . . . 98 26 A c e t y l a t i o n of anthosterone B (119) 99 27 MS fragmentation of 120 109 28 MS fragmentation of compound F 121 114 29 Biosynthetic proposal f o r compounds A and B . . . 116 30 Biosynthetic proposal f o r A-nor steroids 118, 119 118 31 Formation of diosphenols 120, 121 119 32 Biosynthetic proposal f o r shortened side chains 119 - X -LIST OF APPENDICES Appendix 1 Bioassay r e s u l t s f o r phidolopin (36) and desmethylphidolopin (37) 139 x i -ACKNOWLEDGEMENTS I would l i k e to express my appreciation to Professor Raymond J . Andersen f o r h i s encouragement and guidance throughout the course of t h i s work, and for h i s assistance during the preparation of t h i s t h e s i s . Also, I wish to thank a number of people who have a s s i s t e d me i n the c o l l e c t i o n of the organisms studied, e s p e c i a l l y Mr. Mike LeBlanc. I thank Drs. G.K. Eigendorf and S.O. Chan and t h e i r s t a f f f o r e f f i c i e n t assistance and cooperation i n the c o l l e c t i o n of spectroscopic data. F i n a l l y , I wish to extend a very s p e c i a l thanks to my parents f o r t h e i r patience, constant encouragement and support throughout the course of my studies. - x i i -ABBREVIATIONS CDCI3 - Chloroform-CHCI3 = Chloroform DMSO - Dimethylsulfoxide EtOAc - Ethyl acetate g - Grease peak HPLC - High performance l i q u i d chromatography HRMS - High r e s o l u t i o n mass spectrum IR - Infrared MS - Low r e s o l u t i o n mass spectrum Na2S04 - Sodium s u l f a t e (anhydrous) •^H NMR - Proton nuclear magnetic resonance NMR - Carbon-13 nuclear magnetic resonance NOE - Nuclear Overhauser enhancement mp - Melting point RT - Room temperature S - Solvent s i g n a l SCUBA - S e l f contained underwater breathing apparatus TLC - Thin layer chromatography U - Unknown impurity s i g n a l UV - U l t r a v i o l e t - x i i i -Abbreviations f o r m u l t i p l i c i t i e s of NMR s i g n a l s : s = s i n g l e t d = doublet t = t r i p l e t q = quartet dd = doublet or doublets bs = broad s i n g l e t m - m u l t i p l e t This thesis has been written i n conformance with the "Handbook for Authors", published by the American Chemical Society; Washington, D.C., 1978. - 1 -A. INTRODUCTION TO THE BRYOZOANS Bryozoans, commonly found i n abundance i n predator-rich, competi-t i v e environments, have demonstrated the a b i l i t y to grow on a l l forms of hard surface e c o l o g i c a l space such as rocks, s h e l l s , c o r a l s , wood and s t e e l . The a d a p t a b i l i t y of bryozoans has made them one of the most prevalent groups of f o u l i n g organisms. For example, some 130 species have been taken from ship bottoms, where they show a general resistance to a n t i f o u l i n g p a i n t s . ^ Their remarkable adhesion properties have l e d to f u l l scale studies on t h e i r secretory products i n the hope of f i n d i n g v i a b l e new bioadhesive materials acceptable f o r c l i n i c a l use by the dental and medical professions.^ Of p a r t i c u l a r i n t e r e s t , however, are the chemical studies conducted on marine bryozoans i n the past twenty years which have y i e l d e d a number of novel b i o l o g i c a l l y - a c t i v e secondary metabolites. These findings have prompted continuing chemical studies of members of t h i s phylum which have as t h e i r goals the discovery of new b i o l o g i c a l l y - a c t i v e compounds and answers regarding the o r i g i n of the i s o l a t e d metabolites. I. Biology The phylum Bryozoa (or polyzoa or Ectoprocta) contains approxi-mately 4000 known l i v i n g species. The phylum i s divided into three classes, Phylactolaemata, Gymnolaemata, and Stenolaemata. The classes - 2 -Stenolaemata and Gymnolaemata include only marine bryozoans while the class Phylactolaemata i s r e s t r i c t e d to approximately 50 known fresh water species. Bryozoans are c o l o n i a l f i l t e r feeding animals which vary i n height and width and occur i n a v a r i e t y of morphological forms. Some of the names used to describe these forms are f a l s e - c o r a l s , sea-mats, and moss - animals. In B r i t i s h Columbia a l l of these forms are common. Some examples include: Heteropora p a c i f i c a - c o r a l l i k e , Bugula sp. - moss-animal type, and Membranipora membranacea - sea-mat type. Bryozoan colonies are b u i l t of a r e p l i c a t e d s e r i e s of i n d i v i d u a l zooids which have body walls which are calcareous, gelatinous or chitinous and us u a l l y about 0.5 mm i n length. Individuals of most species are encased i n a no n - l i v i n g envelopment that contains an opening ( o r i f i c e ) f o r the protrusion of the c i r c u l a r or horse-shoe shaped lophophore used to gather the small plankton ( c h i e f l y diatoms and other phytoplankton) that make up the bryozoans' d i e t . The i n t e r i o r of the body i s occupied l a r g e l y by the spacious coelom, a U shaped dig e s t i v e t r a c t , muscles and the anus which opens alongside the lophophore. The zooids of a colony are attached through pores or gaps i n the body wall. Some zooids i n a colony are modified for s p e c i a l i z e d functions (poly-morphism) such as feeding (autozooid, the la r g e s t zooid), cleaning, p r o t e c t i o n or brooding of the young (heterozooids). Bryozoan colonies are hermaphroditic with both male and female zooids occurring i n the same colony. In most species, the f e r t i l i z e d egg w i l l pass into a brooding chamber producing larvae which are non-feeding. The larvae have a very b r i e f planktonic existence before - 3 -s e t t l i n g on a hard surface. Non brooding bryozoans possess larvae with a f u n c t i o n a l d i g e s t i v e t r a c t which can feed during the several months of l a r v a l l i f e p r i o r to s e t t l i n g . A f t e r settlement, the larvae begins to transform into a zooid within hours. I i ) Natural Products Chemistry The b i o l o g i c a l and e c o l o g i c a l aspects of bryozoans have been exhaustively investigated, however, u n t i l recent years very l i t t l e work had been conducted on the chemistry of bryozoans. The f i r s t modern chemical and pharmacological studies of marine invertebrates were c a r r i e d out around 1960, with extracts from sponges 3 and coelenterates^ showing some i n t e r e s t i n g a n t i b i o t i c a c t i v i t y . In 1970, P e t t i t and Day reported the f i r s t evidence of a n t i n e o p l a s t i c a c t i v i t y i n extracts of marine i n v e r t e b r a t e s . 5 Roughly 9-10% of the organisms tested displayed a s i g n i f i c a n t l e v e l of a c t i v i t y against the U.S. National Cancer I n s t i -tutes (NCI) murine P388 lymphocytic leukemia (PS system) or Walker carcinosarcoma 256 t e s t l i n e s i n the r a t . Among the organisms tested by P e t t i t et a l . was Bugula n e r i t i n a (bryozoa), c o l l e c t e d o f f the Northeast coast of the Gulf of Mexico. The anticancer studies i n i t i a t e d i n 1968 by P e t t i t et a l . on B. n e r i t i n a culminated i n 1982 with the i s o l a t i o n and structure e l u c i d a t i o n of a remarkable a n t i n e o p l a s t i c compound named b r y o s t a t i n 1 (1).^ The structure of t h i s compound, which was the f i r s t pure metabolite i s o l a t e d from a bryozoan, was solved by a combination of x-ray c r y s t a l l o g r a p h i c 4a-and spectroscopic techniques. B r y o s t a t i n 1 (1) was act i v e against the murine P388 lymphocytic leukemia (PS system) both i n vivo and i n v i t r o . Subsequent studies on the sea-mat l i k e B^ . n e r i t i n a have y i e l d e d four s t r u c t u r a l v a r i e n t s on the same bryopyran macrolide skeleton (la) found i n b r y o s t a t i n 1 (1). They are bryostatins 2 (2) 7 , 3 (3) 8 , 4 ( 4 ) 9 , 5 ( 5 ) 1 0 , 6 ( 6 ) 1 1 and 7 ( 6 a ) . 1 0 R = H o 1a R ' = H 4 -4b-- 5 -5 R= C O B u , R= A c 6 R= C O P r n , R= Ac 6a R = C O C H 3 , R = H 7 R'= R"= C O P r n A simultaneous study by P e t t i t et a l . on Amanthia convoluta (bryozoan) also y i e l d e d an extract possessing a n t i n e o p l a s t i c a c t i v i t y . A. convoluta. which was found to grow together with B^ . n e r i t i n a i n a p a r a s i t i c or e p i p h y t i c - l i k e manner, has y i e l d e d the same group of bryos-t a t i n s (1-6) as well as a new compound designated b r y o s t a t i n 8 ( 7 ) . ^ I t i s unclear whether b r y s t a t i n 8 i s a genuine constituent of A.  convoluta or t r a n s f e r r e d from JL. n e r i t i n a and concentrated by A.  convoluta. - 6 -S t i l l to be determined i s whether these compounds or i g i n a t e from de novo biosynthesis or from a common food source such as b a c t e r i a or phytoplankton. P e t t i t has postulated that the i s o l a t i o n of b r y o s t a t i n 4 (4) from n e r i t i n a c o l l e c t e d i n such diverse geographical areas as the Gulf of Mexico and the Gulf of Sagami, Japan indicates that the macrocy-c l i c lactones are biosynthetic products rather than of dietary o r i g i n . However, t h i s can only be answered by f i n d i n g a di e t a r y source or by biosy n t h e t i c 1 4 C - a c e t a t e feeding s t u d i e s . 1 3 Christophersen and Carle, i n a serie s of a r t i c l e s s t a r t i n g i n 1978 reported the i s o l a t i o n of monoterpenes and nine new bromo-alkaloids from the bryozoan F l u s t r a f o l i a c e a . Their work with marine bryozoa was i n i t i a t e d by an i n t e r e s t i n the study of chemical messengers. 1 4 In a p r i o r study c a r r i e d out by Al - O g i l y and Knight-Jones 1^ on marine 'transmitters', i t was reported that older fronds of F\_ f o l i a c e a emitted a lemonlike odor. This odor was a t t r i b u t e d to the monoterpene co n s t i t u -ents of the bryozoan, namely, c i s - c i t r a l ( 8 ) , t r a n s - c i t r a l ( 9 ) , c i t r o n e l l o l (10), ne r o l (11) and geraniol (12). 1 6 8 9 1 0 1 1 1 2 - 7 -The nine novel bromo-alkaloids isolated from F\_ foliacea possess either the physostigmine, indole, or quinoline ring systems. A l l the structures were solved by spectroscopic means. In 1979, Christophersen and Carle reported the isolation and structure elucidation of flustra-mine A (13) and flustramine B (14), both possessing the unprecedented bromophysostigmine skeleton and a rare N-8 y,7-dimethylallyl substitu-ent. 1 7 13 14 Christophersen and Carle, in a continuation of the study of F.  foliacea (L.), reported the isolation and structure elucidation of flustramine C (15), flustraminol A (16) and flustraminol B (17).^ These three structures possess the basic 6-bromo physostigmine skeleton, however, in contrast to compounds (13) and (14) they a l l contain only one isoprene substituent. In 1981, Wulf et a l . reported the isolation - 8 -and structure e l u c i d a t i o n of a bromo-indole d e r i v a t i v e , flustrabromine (18).1^ A d d i t i o n a l J\ f o l i a c e a metabolites include flustramide A (19), 6-bromo-N-methyl-N-formyltryptamine (20)^ u and 7-bromo-4-(2-ethoxy-et h y l ) q u i n o l i n e (21),21 the f i r s t i s o l a t e d n a t u r a l l y occurring bromo-quinoline. A s e r i e s of new strongly a n t i m i c r o b i a l bromoalkaloids, dihydroflustramine C (22), flustramine D (23), dihydroflustramine C N-oxide (24), flustramine D N-oxide (25) and isoflustramine D (26), were recently i s o l a t e d from F l u s t r a f o l i a c e a c o l l e c t e d i n the Bay of Fundy.22 C h a r t e l l i n e A (27), a novel pentahalogenated a l k a l o i d from the bryozoan C h a r t e l l a papyracea. which belongs to the same family as F.  f o l i a c e a . was reported i n 1985 by Chevolot et al.23 The structure of c h a r t e l l i n e A (27) was unambiguously assigned by s i n g l e c r y s t a l X-ray crystallography. 10 -- 11 -Two gramine derived bromo-alkaloids, 2 ,5 ,6-tribromo-N-methylgramine (28) and i t s N-oxide (29), were i s o l a t e d by Sato and F e n i c a l i n 1983 from the subtropical bryozoan Zoobotryon v e r t i c i l a t u m . 2 4 A l k a l o i d N-oxides are commonly found along with free a l k a l o i d s from t e r r e s t r i a l sources, however, t h e i r occurrence i n F\. f o l i a c e a 2 2 and Z^ . v e r t i c i l a t u m 2 ' 0 29 12 -appear to be the only such reported examples from marine sources. I n i t i a l bioassays show that compound (28) i n h i b i t s c e l l d i v i s i o n of f e r t i l i z e d sea urchin eggs (ED50 =16 ug/mL). Compound (28) was synthesized by an I t a l i a n group i n 1977.^ 5 (2-Hydroxyethyl)dimethylsulfonium ion (30), has been determined to be the causative agent of Dogger Bank i t c h , an eczematous a l l e r g i c contact dermitis caused by exposure to the bryozoan Alcyonidium  gelatinosum.^° Sulfoxonium ions had not previously been found i n nature. 0" C H 3 I C H 3 / * C H 2 — C H 2 — OH 30 Nudibranchs are one of the main bryozoan predators. Methanolic extracts of three nudibranchs, Roboastra t i g r i s . Tambje e l i o r a and Tambi e abdere. have a l l been found to contain the same group of b i o l o g i c a l l y a c t i v e b i p y r r o l e s , tambjamines A-D (31-34).^ These compounds were a l l traced to a dietary source, the bryozoan Sessibugula  translucens. Tambjamines A-D are the major secondary metabolites of S.  translucens and i t i s believed the nudibranchs use these dietary compounds f o r s e l f defence. When FL. t i g r i s . the large carnivorous 13 -33 34 nembrothid nudibranch attacks the two smaller nembrothid nudibranchs T. abdere and Tj. e l i o r a the following occurs; T\_ abdere produces a yellow mucus from goblet c e l l s i n the skin which causes R^ . t i g r i s to break o f f the attack. The sec r e t i o n has been shown to contain mainly tambjamines A-D. Tj_ e l i o r a does not appear to produce a defensive secretion, however, i t d i d attempt to escape t i g r i s by a vigorous writhing motion. In subsequent laboratory observations i t has been - 14 -shown that |L_ t i g r i s preferred to eat Tj. e l i o r a rather than L abdere. In further bioassay studies, these b i p y r r o l e s have displayed antimicro-b i a l a c t i v i t y against various b a c t e r i a . Fusetani et a l . 2 8 recently reported the i s o l a t i o n of a blue a n t i -m i c r o b i a l t e t r a p y r r o l e 35 from the bryozoan Bugula dentata. Compound 35 was previously found i n a mutant s t r a i n of the b a c t e r i a S e r r a t i a  marcescens. A new purine d e r i v a t i v e , phidolopin (36), was reported In 1984 by Ayer and Andersen 2 9 from the bryozoan Phidolopora p a c i f i c a c o l l e c t e d i n the waters o f f B r i t i s h Columbia. Co-occurring with t h i s compound was i t s desmethyl d e r i v a t i v e (37), as well as two nitrophenols, 4-hydroxy-methyl-2-nitrophenol (38) and 4-methoxymethyl-2-nitrophenol (39). Phidolopin was shown to possess potent i n - v i t r o a n t i m i c r o b i a l and a n t i -a l g a l a c t i v i t i e s . - 1 5 -- 16 -B. SYNTHESIS OF PHIDOLOPIN AND DESMETHYLPHIDOLOPIN The t o t a l s y n t h e s i s of p h i d o l o p i n (36) and desmethylphidolopin (37) was undertaken i n order to confirm the s t r u c t u r e of 37 proposed by Ayer 9 9 et a l . * i n t h e i r 1984 study of the chemistry of Pj. p a c i f i c a . and a l s o to devise an e f f i c i e n t method f o r producing s u f f i c i e n t q u a n t i t i e s of both m e t a b o l i t e s f o r f u r t h e r b i o l o g i c a l t e s t i n g . The s y n t h e t i c p l a n f o r the t o t a l s y n t h e s i s of these two compounds i s shown i n Scheme 1. The f i r s t step o f the s y n t h e s i s e n t a i l e d the p r o t e c t i o n o f the hydroxyl f u n c t i o n a l i t y on 4-methyl - 2-nitrophenol (40) w i t h a base s t a b l e p r o t e c t i n g group, which could be e a s i l y removed as the f i n a l step i n s y n t h e s i s . P r o t e c t i n g the phenol as i t s methyl ether or as i t s methoxymethyl ether (MOM) appeared to be the most d i r e c t approach. The f o l l o w i n g methods were attempted to accomplish the d e s i r e d p r o t e c t i o n i n the grea t e s t y i e l d (Scheme 2). M e t h y l a t i o n of 4-methyl -2-- 17 ieme 1: Synthetic plan f o r phidolopin (36) and desmethyl-phidolopin (37) 36 R=CH3 37 R=H 18 -Scheme 2: Protection of 4-methyl-2-nitrophenol 40 a b c 40 nitrophenol (40) was achieved by reacting 40 i n acetone and excess potassium carbonate with 1.5 equivalents of methyl iodide under r e f l u x (Scheme 2a).^ u Continuous monitoring by t h i n layer chromatography (TLC) (1:10 e t h y l acetate/petroleum ether) showed rapid formation of a new UV absorbing component at Rf 0.13 which was more polar than the s t a r t i n g material at Rf 0.35. A f t e r 2.5 hr, i t appeared by t i c that the reaction - 19 had proceeded to completion. Work-up of the r e a c t i o n mixture by p a r t i -t i o n i n g between d i s t i l l e d water and dichloromethane, drying of the organic layer with Na2S0^, f i l t e r i n g and evaporation i n vacuo y i e l d e d a heavy brown o i l . P u r i f i c a t i o n of t h i s o i l by preparative TLC (2:10 e t h y l acetate/petroleum ether) gave 41 as a viscous yellow o i l ( y i e l d 71%). NMR (Figure 1) and mass spectrometric analysis of t h i s o i l showed the required NMR resonance f o r the OCH3 protons at 6 3.95 ppm (s, 3H), (Table 1) and a MS parent ion at m/z 167 (Scheme 3). Two methods, both i n v o l v i n g the use of chloromethyl methyl ether, but using two d i f f e r e n t bases, were explored as approaches to the preparation of the -OCH2CH3 (MOM) d e r i v a t i v e (Scheme 2b, c ) . 3 1 , 3 2 In the f i r s t case, the phenoxide of nitrophenol (40) was prepared i n dry d i e t h y l ether using a suspension of sodium hydride (excess). Chloro-methylmethylether (1.5 equiv) was added dropwise at 0°C to the red s o l u t i o n of the phenoxide. The reaction, which was c o n t i n u a l l y moni-tored by TLC (1:10 e t h y l acetate/hexanes), was allowed to proceed at room temperature for 48 hr. A new more polar UV absorbing component (Rf 0.17) was formed. The work-up involved p a r t i t i o n i n g the r e a c t i o n mixture between water and d i e t h y l ether, drying the organic layer over Na2S04, f i l t e r i n g and evaporation i n vacuo. P u r i f i c a t i o n by preparative TLC (1:10 e t h y l acetate/hexanes) y i e l d e d 42 as a c l e a r yellow o i l ( y i e l d 36.3%). The -^H NMR of 42 (Figure 2) showed resonances at 6 3.53 ppm (s, 3H), and 6 5.25 ppm (s, 2H) assigned to the MOM group (Table 1). A EIMS of 42 showed a parent ion at m/z 197 as required (Scheme 4). A large amount of unreacted s t a r t i n g material 40 was also recovered from this r e a c t i o n . - 22 -Table 1: 80 MHz iH NMR data on protected nitrophenols 41 and 42 Chemical s h i f t , 6 ppm (CDCI3) H on C # 41 42 3 7.65 (d, J - 2 Hz, IH) 7.56 (d, J - 2 Hz, IH) 5 7.32 (dd, J = 9, 2 Hz, IH) 7.30 (dd, J = 9, 2 Hz, IH) 6 6.97 (d, J •= 8 Hz, IH) 7.13 (d, J •= 9 Hz, IH) 7 2.35 (s, 3H) 2.35 (s, 3H) 8 3.95 (s, 3H) 5.25 (s, 2H) 9 3.53 (s, 3H) 23 Scheme 3: Interpretation of the MS fragmentation of 41 m/z 105 (27°/ 0) 0 C H 3 " 1 * # / ^ N 0 2 / - C H 3 NO 2 -NO m/z 137 (49 •/.) 41 m/z 167(78%) U02 m/z 120 ( 82 %) Scheme 4: Interpr e t a t i o n of the MS fragmentation of 42 —I" 0CH,0CH 3 I m / z '3&<t' X) — » m/z 167 ( 37 %) OCH: 42 m/z 166(3%) m / z 197( 55%) - 24 -In an attempt to devise a higher y i e l d i n g and cleaner method for the preparation of the MOM de r i v a t i v e 42, we modified the reaction conditions to use the milder base, potassium carbonate. Thus, excess potassium carbonate and 6 equivalents of chloromethyl methyl ether were reacted with the nitrophenol 40 i n acetone at room temperature. Under these conditions, the rea c t i o n went almost spontaneously upon addi t i o n of the chloromethyl methyl ether as indicated by TLC (1:10 eth y l ace-tate/hexanes). There appeared to be much les s s t a r t i n g material a f t e r 10 to 15 minutes than there was i n the previous method a f t e r 48 hr. Following work-up and p u r i f i c a t i o n conducted as before, a s i n g l e compo-nent ( y i e l d 73%) was i s o l a t e d , which had i d e n t i c a l s p e c t r a l features to the previously prepared MOM protected nitrophenol 42. L i s t e r et a l . 3 3 have looked at the optimization of conditions f o r production of 7-benzylxanthine (43) and 9-benzylxanthine (44) deriva-t i v e s v i a the N7 and N9 a l k y l a t i o n of xanthines. 0 43 44 They found that 7-(4-nitrobenzyl)theophylline (48) or 7-(4-methyl-benzyl)theophylline (50) could be produced by r e f l u x i n g 4-nitrobenzyl-- 25 -bromide (47) or 4-methylbenzylbromide (49) with theophylline (45) i n aqueous sodium hydroxide (.1 M) for 2 hours (Schemes 5 and 6). Using L i s t e r ' s work as a model, i t seemed that a l o g i c a l next step i n the synthesis of phidolopin (36) and desmethylphidolopin (37) was the conversion of the protected nitrophenols 41 and 42 to benzyl bromide de r i v a t i v e s which could act as a l k y l a t i n g agents. Bromination of 4-methoxy-3-nitrotoluene (41) was s u c c e s s f u l l y c a r r i e d out with N-bromosuccinamide (NBS) as the brominating agent (Scheme 7 ) . 3 4 The r e a c t i o n was c a r r i e d out by d i s s o l v i n g compound 41 and NBS (2 equiv) i n a minimum amount of d i s t i l l e d carbon t e t r a c h l o r i d e and then r e f l u x i n g t h i s s o l u t i o n i n the presence of l i g h t (150 W tungsten bulb). Scheme 5: A l k y l a t i o n of theophylline (45) with 47 Base Br 45 47 48 26 -Scheme 6: A l k y l a t i o n of theophylline with 49 - 27 The r e a c t i o n was monitored very c l o s e l y by TLC (3:10 eth y l acetate/ hexanes) to ensure that only monobromination occurred. A f t e r only 5 minutes, a new more polar component became apparent on TLC. As time proceeded, the reac t i o n mixture went from yellow to a reddish brown, perhaps i n d i c a t i n g the formation of bromine. When a s u f f i c i e n t amount of the new component had formed, the reaction was halt e d and c a r e f u l l y worked up. Rapid p u r i f i c a t i o n of the new component by preparative TLC (3:10 eth y l acetate/hexanes) i n a darkened room, afforded a yellow o i l ( y i e l d 45%), which was i d e n t i f i e d by low r e s o l u t i o n mass spectrometry to be the desired benzyl bromide 51 (Scheme 8). NMR was not use f u l i n the c h a r a c t e r i z a t i o n of 51 due to the rapid decomposition of t h i s highly l a b i l e substance. Scheme 8: MS fragmentation of compound 51 (25 V.) * m/z 166 (657.) 51 m/z 245/ 247(2,3 V.) - 28 -The bromination of the MOM protected nitrophenol 42 was c a r r i e d out i n a s l i g h t l y modified manner (Scheme 9). Compound 42 was dissolved i n a minimum amount of carbon t e t r a c h l o r i d e and the s o l u t i o n was brought to r e f l u x while being i r r a d i a t e d with a 220 W sun lamp. Two equivalents of NBS i n carbon t e t r a c h l o r i d e were slowly added to the r e f l u x i n g s o l u t i o n . The r e a c t i o n was c a r e f u l l y monitored by TLC (3:10 eth y l acetate/hexanes) to ensure that only monobromination occurred. Scheme 9 : Bromination of MOM protected nitrophenol 42 0M0M 0M0M When the rea c t i o n was l e f t to run f o r more than 20 minutes, the forma-t i o n of polybrominated species would occur as indi c a t e d by the appear-ance of numerous more polar TLC spots. The monobrominated product 52 was v i s i b l e on TLC within 5 minutes and the rea c t i o n was complete by 15 minutes. The r e a c t i o n was terminated by removing the l i g h t source. To avoid the loss of material due to decomposition, the brominated species was never i s o l a t e d f o r cha r a c t e r i z a t i o n , but instead i t was used d i r e c t l y i n the a l k y l a t i o n reactions. Even though 52 was not character-- 29 -ized s p e c t r o s c o p i c a l l y , TLC showed 52 to be s l i g h t l y more polar (Rf 0.28) than the s t a r t i n g material 42 (R f 0.34) (3:10 eth y l acetate/hexanes) s i m i l a r to the p o l a r i t y differences between 41 and 51. A l k y l a t i o n was c a r r i e d out with both the methyl as well as the MOM protected a l k y l a t i n g agents. Reaction of 51 with theophylline i n 1.5 mL of r e f l u x i n g 0.1 M sodium hydroxide and 3 mL tetrahydrofuran f o r 2 hr gave both the desired N7 a l k y l a t e d compound 53 as well as the N9 a l k y l a t e d compound 54 (Scheme 10). The N7 and N9 a l k y l a t e d products could be distinguished by examination of the NMR spectra of the two compounds (Table 2). The chemical s h i f t of the benzylic protons i n the Scheme 10: A l k y l a t i o n of theophylline (45) with 51 - 30 -Table 2: 80 MHz iH NMR data comparison of 53 and 54 Chemical s h i f t , 6 ppm H on C or N # 53 54 1' 5. .48 (s, 2H) 5. .53 (s, 2H) NI Me 3. .43 (s, 3H) 3. .39 (s, 3H) N3 Me 3. .58 (s, 3H) 3. .56 (s, 3H) 3' 7, .38 (dd, J •= 9, 2 Hz, IH) 7, .58 (dd, J - 9, 2 Hz, IH) 4' 7 .00 (d, J -= 9 Hz, IH) 7, .09 (d, J = 9 Hz, IH) 7' 7. .68 (d, J • 2 Hz, IH) 7 .89 (d, J •= 2 Hz, IH) 4' NO - 31 -N7 a l k y l a t e d species was found to be 6 5 .48 ppm (s, 2H), whereas the benzylic protons i n the N9 a l k y l a t e d compound resonated at 6* 5 .53 ppm (s, 2H). Also a l t e r e d were the s h i f t s of the aromatic protons as seen i n Table 2. Comparison of the chemical s h i f t s of the be n z y l i c protons to the observed values f o r phidolopin (36) allowed the assignment of the correct structures. In the case of the MOM d e r i v a t i v e 42, the bromination and a l k y l a -t i o n reactions were c a r r i e d out without I s o l a t i o n of intermediate 52 (Scheme 11). Following the bromination reaction, the r e a c t i o n mixture was b r i e f l y cooled and the succinamide was f i l t e r e d o f f . Scheme 11: Bromination and a l k y l a t i o n 5 5 R=CH 56 R=H 32 -The r e s u l t i n g f i l t r a t e was c a r e f u l l y evaporated i n vacuo to near dryness before being dissolved i n THF and added dropwise to a s o l u t i o n of theophylline (45) i n 0.1 M sodium hydroxide s o l u t i o n which had been s t i r r i n g f o r 20 minutes at room temperature. The amount of theophylline used i n the a l k y l a t i o n reaction assumed a 100% y i e l d i n the bromination reaction. As a r e s u l t , excess theophylline (45) always appeared i n the r e a c t i o n mixture. A f t e r 18 hours of s t i r r i n g at room temperature, TLC analysis showed the presence of the protected nitrophenol 42 as well as a new more polar component. Work-up and p u r i f i c a t i o n by preparative TLC (3:10 e t h y l acetate/hexanes) y i e l d e d the polar constituent as a white s o l i d ( y i e l d 25%) which was shown by ^H NMR (Figure 3) and mass spectrometric analyses to be the desired protected phidolopin 54. Key features of the ^H NMR spectrum were a sharp two proton s i n g l e t at S 5.46 ppm assigned to benzylic protons and resonances at S 3.51 and 5.25 ppm assigned to the MOM group. The mass spectrum of 55 showed a parent ion at m/z 375 (8% of base) (Scheme 12). The y i e l d i n t h i s two step r e a c t i o n (32%) proved to be the lowest i n the t o t a l synthesis, however, nearly 50% of the protected nitrophenol 42 was recoverable by chromato-graphic means a f t e r work-up of the reaction. Using the same method of bromination and a l k y l a t i o n , MOM protected desmethylphidolopin (56) was synthesized i n nearly the same y i e l d (24.7%) by s u b s t i t u t i n g 3-methylxanthine (46) f o r theophylline (45) (Scheme 11). As before, only the N7 product was formed, with no e v i -dence of NI or N9 a l k y l a t i o n (Figure 4) . L i s t e r 3 - * found that exclusive N9 a l k y l a t i o n of xanthines could be achieved by carrying out the a l k y l a -t i o n i n a more concentrated basic s o l u t i o n under r e f l u x conditions for 3 Figure 4: 80 MHz iH NMR of MOM protected desmethylphidolopin 56 35 -Scheme 12: MS fragmentation of 55 hours while C-8 a l k y l a t i o n would occur i n dimethylformamide under r e f l u x f o r 4 hours (Scheme 13). I t appears that the N7 a l k y l a t i o n product i s the k i n e t i c a l l y preferred product while the N9 and C8 are the thermo-dynamically preferred a l k y l a t i o n products of theophylline. As seen previously i n the a l k y l a t i o n of theophylline with the methylated nitrophenol 41, r e f l u x conditions y i e l d e d a mixture of products (Scheme 9). Therefore, i t appears the use of milder r e a c t i o n conditions pro-motes the a l k y l a t i o n of only one p o s i t i o n , N7. Deprotection of the methylated phidolopin d e r i v a t i v e 53 proved to be d i f f i c u l t . Using the method outlined by Olah et a l . , 3 ^ which allows fo r the cleavage of methyl ethers by treatment with chlorotrimethyl-- 36 -Scheme 13: A l k y l a t i o n of theophylline (45) under v a r i e d conditions s i l a n e and sodium iodide i n dry a c e t o n i t r i l e at room temperature f or 8 to 10 hours, y i e l d e d only s t a r t i n g material (Scheme 14). Deprotection of the MOM der i v a t i v e s proved to be much simpler and gave high y i e l d s of product. Refluxing e i t h e r MOM d e r i v a t i v e 55 or 56 for one hour i n a minimum amount of chloroform containing 50% ac e t i c a c i d 3 7 plus one drop of concentrated sulphuric a c i d y i e l d e d phidolopin - 37 -Scheme 14: Deprotection of 53 with chlorotrimethylsilane (36) and desmethylphidolopin (37) r e s p e c t i v e l y (Table 3) a f t e r p u r i f i c a t i o n by preparative TLC (Figures 5, 6 ) (Scheme 15). Scheme 15: Deprotection of 55 and 56 with d i l u t e a c i d 55 R=CH 3 56 R=H 36 R=CH3 37 R=H Figure 5: 80 MHz 1H NMR of phidolopin 36 - 40 -Table 3: 80 MHz -^H NMR data f o r phidolopin (36) and desmethyl-phidolopin (37) Chemical s h i f t , 5 ppm H on C or N # 36 37 NI--H 11.03 (s, IH) NI Me 3. ,59 (s, 3H) -N3 Me 3. 59 (s, 3H) 3.46 (s, 3H) 8 7. .65 (s, IH) 7.82 (s, IH) 1' 5. .48 (s, 2H) 5.45 (s, 2H) 3' 7. .63 (dd, J-l.9,8.5 Hz, IH) 7.68 (dd, J-8.5,1.9 Hz, IH) 4' 7, .16 (d, J - 8.5 Hz, IH) 7.10 (d, J - 8.5 Hz, IH) 7' 8 .06 (d, J = 1.9 Hz,' IH) 8.05 (d, J = 1.9 Hz, IH) 36 37 - 41 -Spectroscopic data (Table 3) obtained f o r the two synthetic products was i d e n t i c a l to the spectroscopic data recorded f o r the natural products. The t o t a l synthesis of phidolopin (36) was reported o o by a Japanese group J O simultaneously with the completion of the t o t a l syntheses of phidolopin (36) and desmethylphidolopin ( 3 7 ) 3 ^ by t h i s author i n 1985. In addition to the synthesis of compound (36), assignment of the 1 3 C NMR spectrum was reported (Table 4 ) . Using t h i s data and NMR data of compound (36) (Figure 7), assignment of the spectrum of desmethylphidolopin (37) (Figure 8) was s i m p l i f i e d (Table 4 ) . - 44 -Table 4: 1 J C NMR data comparison f o r 3 6 j e and 37 Chemical s h i f t , 5 ppm C # 36 a 37 b NI Me 28. 0 (q) -N3 Me 29. 7 (q) 28. 5 (q) 2 155. ,3 (s) 154. ,9 (s) 4 149. .3 (s) 150. .4 (s) 5 106. .8 (s) 106. .3 <s) 6 151. .6 (s) 151, .1 (s) 8 140. .6 (d) 142, .3 (d) 1' 49. .0 (t) 47, .9 (t) 2» 128 .1 (s) 127, .6 O) 3' 124 .5 or 121.0 (d) 119 .9 or 125.2 (d) 4' 133. .7 (s) 135, .2 (s) 5' 155, .2 (s) 152, .8 (s) 6' 124. .5 or 121.0 (d) . 119, .9 or 125.2 (d) 7' 137. .0 (d) 136. .3 (d) a b 1 0 0 MHz, C D C 1 3 75 MHz, C D C I 3 + D M S 0 - d 6 - 45 -C. NITROPHENOLS FROM NORTHEAST PACIFIC BRYOZOANS The i s o l a t i o n of phidolopin (36), from the bryozoan Phidolopora  p a c i f i c a c o l l e c t e d o f f the coast of B r i t i s h Columbia, was reported by Ayer et al.^9 i n 1984. Attention was drawn to t h i s bryozoan by the lack of f o u l i n g organisms on i t s skeleton as well as by the strong i n v i t r o a n t i - a l g a l and a n t i - b a c t e r i a l a c t i v i t y displayed by i t s crude extracts. Further i n v e s t i g a t i o n of P_j_ p a c i f i c a ( r e f e r r e d to as the "lacy bryozoan" due to i t s i n t r i c a t e calcium carbonate exoskeleton which resembles a r u f f l e d l a c y network) l e d to the i s o l a t i o n of other novel nitrophenols; desmethylphidolopin (37), 4-hydroxymethyl-2-nitrophenol (38) and 4-methoxymethyl-2-nitrophenol (39), the l a t e r b e l i e v e d to be an i s o l a t i o n a r t i f a c t . Encouraged by the discovery of i n t e r e s t i n g b i o l o g i c a l l y a c t i v e secondary metabolites i n Pj. p a c i f i c a . we i n i t i a t e d an examination of other common Northeastern P a c i f i c bryozoans which l i v e i n s i m i l a r habitats with the hope of discovering a d d i t i o n a l new b i o l o g i c a l l y active metabolites. A l l new bryozoan specimens were c o l l e c t e d by hand using SCUBA. Crude methanol extracts of these animals were assayed for i n -v i t r o a n t i b a c t e r i a l , antifungal and a n t i a l g a l a c t i v i t y . Diaperoecia c a l i f o r n i c a (d'Orbigny 1852), u s u a l l y r e f e r r e d to as the "Southern Staghorn" bryozoan, i s commonly found on offshore reefs as well as i n t e r t i d a l rocks (-2 to -10 m) from the B r i t i s h Columbia coast to Baja, C a l i f o r n i a . C l a s s i f i e d i n the bryozoan order Cyclostomata, D.  c a l i f o r n i c a possesses an i n f l e x i b l e calcium carbonate exoskeleton which - 46 -i s c o r a l - l i k e i n appearance, with each colony a t t a i n i n g a height of approximately 10 cm and a diameter of 13 to 15 cm. I t i s also i d e n t i -f i e d by i t s l i g h t yellow tubular branches which have a f l a t cross-s e c t i o n . D. c a l i f o r n i c a was f i r s t c o l l e c t e d i n June 1984 i n Barkley Sound, B.C. The bryozoans (653 g drie d weight a f t e r extraction) were immedi-at e l y soaked i n methanol and r e f r i g e r a t e d (-2°C) for up to seven days before work-up. At the end of t h i s t ime, the methanol layer was decanted o f f and the bryozoans were ground i n a waring blender with - 47 fresh methanol, and f i l t e r e d . The combined reddish-brown methanol extracts were concentrated i n vacuo down to about one t h i r d the o r i g i n a l volume and the r e s u l t i n g aqueous methanolic layer was extracted with e t h y l acetate. The red-brown ethyl acetate soluble extract was d r i e d over anhydrous sodium s u l f a t e . The sodium s u l f a t e was f i l t e r e d o f f and the e t h y l acetate was evaporated i n vacuo to y i e l d 5.8 g (.89%) of a dark red o i l . Flash chromatography gave three f r a c t i o n s d i s t i n g u i s h a b l e by s i m i l a r chromatographic p o l a r i t i e s , each of which on bioassay analy-s i s afforded moderate antifungal and a n t i b a c t e r i a l a c t i v i t y . P u r i f i c a -t i o n of the most polar f r a c t i o n by column chromatography on Sephadex LH-20 (7:3 methanol/chloroform) gave a strongly retained yellow band. TLC a n a l y s i s of t h i s band indicated a s i n g l e component with an Rf value s i m i l a r to phidolopin (36), which strongly absorbed both short wave (dark) and long wave (black) UV l i g h t . Analysis by *H NMR (Table 5) as well as mass spectrometry (Scheme 16) confirmed the presence of phidolo-p i n (36) (1.0 mg .001%), as yellow needles (mpt. 211-212). P u r i f i c a t i o n of the two l e s s polar f r a c t i o n s by column chromato-graphy on Sephadex LH-20 (7:3 methanol/chloroform) gave desmethyl-phidolopin (37) (3.7 mg, .001%) as a yellow s o l i d (Table 5) (Scheme 17), as w e l l as 4-hydroxymethyl-2-nitrophenol (38) (4.5 mg, .007%) as a yellow o i l (Table 6) (Scheme 18). The 1H NMR and mass spectra of the compounds were i d e n t i c a l to the spectra of authentic materials. This general i s o l a t i o n and p u r i f i c a t i o n procedure was employed i n the examination of three other bryozoans, namely, Heteropora alaskensis. Hippodinlosla i n s c u l p t a . and T r i c c e l a r i a ternata with the r e s u l t s tabulated i n Table 13 (Section 6). 1H NMR data (Figures 5, 6, 9, 10) as - 48 -well as MS fragmentation patterns f o r the i s o l a t e d metabolites can be observed i n Tables 5 and 6 and Schemes 16 and 19 r e s p e c t i v e l y . Table 5: NMR data f o r i s o l a t e d nitrophenols 36-37 Chemical s h i f t , S ppm H on C or N # 36 s 37* 1 - -NI Me 3. .40 (s, 3H) -N3 Me 3 .59 (s, 3H) 3, .51 (s, 3H) 8 7, .63 (s, IH) 7. .63 (s, IH) 1' 5 .46 (s, 2H) 5. .39 ( s , 2H) 3' 7. .61 (dd, J-8.5,2.5 Hz, IH) 7, .59 (dd, J - 9,2 Hz, IH) 4' 7 .17 (d, J - 8.5 Hz, IH) 7, .13 (d, J - 9 Hz, IH) 7' 8, .09 (d, J - 2.5 Hz, IH) 8. .06 (d, J - 2 Hz, IH) 80 MHz (CDCl 3-d bDMSO) 49 Scheme 16: MS fragmentation of phidolopin (36) m/z 152 (38 V.) m/z 180 (100 V.) 3 6 m/z 331 ( U V.) Scheme 17: MS fragmentation of desmethylphidolopin (37) • HNCO m/z 166(100 V.) m/z 123 (45 V.) - 50 -Table 6: 1H NMR data f o r i s o l a t e d nitrophenols 38-39 Chemical s h i f t , 6 ppm H on C # 38 39 3 8.09 (d, J = 2 Hz, IH) 8 .10 (d, J = 2 Hz, IH) 5 7.58 (dd, J -= 9, 2 Hz, IH) 7, .60 (dd, J - 8, 2 Hz, IH) 6 7.18 (d, J - 9 Hz, IH) 7, .18 (d, J - 8 Hz, IH) 7 4.69 (s, 2H) 4. .43 (s, 2H) 8 - 3. .44 (s, 3H) ^-OH 10.54 (brs, IH) 10, .58 (s, IH) 38 39 - 53 -Scheme 18: MS fragmentation of nitrophenol 38 OH r ^ N ^ N O o m/z 123 ( 25 V.) N 0 2 • m/z 152 ( 11 V.) 'OH 38 m/z 169 (100 V.) Scheme 19: MS fragmentation of nitrophenol 39 OH V ' ^ 0 C H 3 -0CH 3 t m/z 152 (100 V.) CNO-m/z 136 (21V.) 39 m/z 183 (59V.) - 54 -D. DISCUSSION The i s o l a t i o n of phidolopin ( 3 6 ), desmethylphidolopin ( 3 7 ) as well as nitrophenols ( 3 8 ) and ( 3 9 ) i n some combination from the extracts of f i v e Northeast P a c i f i c bryozoans, which a l l belong to d i f f e r e n t genera, r a i s e s a question about the o r i g i n of these secondary metabolites. 3^ Natural products which contain unusual f u n c t i o n a l i t i e s such as n i t r o groups normally have very r e s t r i c t e d taxonomic d i s t r i b u t i o n s . There-fore, i t seems u n l i k e l y that each member of the diverse group of bryozoans investigated i n t h i s study would elaborate t h i s same group of novel secondary metabolites through de novo biosynthesis. A more probable assumption, that these nitrophenols are obtained by the bryozoans from some sort of dietary or symbiotic microorganism such as a phytoplankter, bacterium or fungus, i s quite consistent with the organisms c h a r a c t e r i s t i c f i l t e r feeding c a p a b i l i t i e s . A survey of other examples of purine d e r i v a t i v e s and n i t r o contain-ing compounds from marine organisms serves as a u s e f u l background for speculation about the o r i g i n of the bryozoan metabolites. Purine d e r i v a t i v e s based on the xanthine nucleus are generally of plant o r i g i n . Phidolopin ( 3 6 ) represents only the second example of a xanthine a l k a l o i d i s o l a t e d from a marine source, the f i r s t being c a f f e i n e ( 5 8 ) , i s o l a t e d from the Chinese gorgonian Echinogorpia  pseudossapo.^ Marine organisms have also elaborated a number of other purine de r i v a t i v e s not based on the xanthine nucleus. Among the most common - 55 -are the purine ribosides such as doridosine ( 5 9 ) . In 1980, Cook et-a l . 4 1 reported the i s o l a t i o n of doridosine (1-methylisoguanosine) from the A u s t r a l i a n marine sponge Tedania d i g i t a t a . This compound was-previously reported from the digestive gland of the dorid nudibranch Anisodoris n o b i l i s a n < j ^ was found to possess potent muscle HO OH 59 - 56 -relaxant, anti-inflanunatory and a n t i - a l l e r g i c a c t i v i t i e s . The i n j e c t i o n of mice with a dose equivalent to lg/Kg of crude sponge extract gave pronounced muscle r e l a x a t i o n and hypothermia which served as a decisive and reproducible assay for p u r i f y i n g 59. Other b i o l o g i c a l l y active marine purine ribosides include spongo-sine (2-methoxyadenosine) ( 6 0 ) 4 3 i s o l a t e d from the sponge Tedania-d i g i t a t a 4 3 and isoguanosine (61), extracted from the dorid nudibranch D i a u l u l a s a n d i e g e n s i s , 4 4 I t i s i n t e r e s t i n g to note that isoguanosine (61) was only found i n sandiegensis c o l l e c t e d from Monterey, C a l i f o r n i a while specimens from La J o l l a , C a l i f o r n i a contained only a s e r i e s of unrelated chlorinated acetylenes. 4-* Adenosine (62) as well as 2'-deoxyadenosine (63) were reported as the c a r d i o a c t i v e constituents of the sponge Dasychalina cyathina F i n a l l y 9-/J-D-arabinosyladenine (64) and i t s 3'-0-acetyl d e r i v a t i v e - 57 -( 6 5 ) , p r e v i o u s l y known as potent s y n t h e t i c a n t i v i r a l agents, have been i s o l a t e d as n a t u r a l products from the I t a l i a n gorgonian E u n i c e l l a  c a v o l i n i . 4 7 Compound 64 was the f i r s t a n t i v i r a l drug used i n the treatment of the u s u a l l y f a t a l herpes e n c e p h a l i t i s . Purine d e r i v a t i v e s other than r i b o s i d e s have a l s o been found from marine sources. Two 9-methyladenine d e r i v a t i v e s of di t e r p e n e s , a g e l i n e A (66 ) and a g e l i n e B ( 6 7 ) , possessing moderate i c h t h y o t o x i c as w e l l as a n t i m i c r o b i a l a c t i v i t y were i s o l a t e d from the P a c i f i c sponge Agelas sp_. 4^ F i v e new 9-methyladenine d e r i v a t i v e s of diterpenes have been i s o l a t e d from the Okinawan sea sponge, Agelas n a k a m u r a i F o u r of these m e t a b o l i t e s , agelasine-A ( 6 8 ) , agelasine-B ( 6 9 ) , agelasine-C (70) - 58 -and agelasine-D (71) are based on b i c y c l i c diterpene skeletons. The f i f t h new compound agelasine-E (72), was reported along with the known compound ageline A (66).^0 These 9-methyladeninium s a l t s i n h i b i t N a +/K +-transporting ATPase i n v i t r o and also possess a n t i m i c r o b i a l and antispasmodic properties. 70 59 -72 Hokupurine (73) has been i s o l a t e d from the nudibranch P h e s t i l l a  melanobranchia as well as the c o r a l Tubastea c o c c i n e a 5 1 upon which i t feeds. In contrast to the other purine d e r i v a t i v e s reported from marine sources, hokupurine (73) has no s i g n i f i c a n t b i o l o g i c a l a c t i v i t y . NH 73 - 60 -This survey of purine metabolites from marine sources shows that nearly a l l the compounds Isolated display some form of s i g n i f i c a n t b i o l o g i c a l a c t i v i t y . In addition, beginning with the f i r s t purine example stated above, caffeine (58), and moving through the compounds of T. d i g l t a t a . A. n o b i l i s . D. sandiegensis and P^ . melanobranchia. i t can be concluded that i n most cases the o r i g i n of the compounds i s not c l e a r . Caffeine (58), f o r example, i s also a well known t e r r e s t r i a l plant natural product. The i s o l a t i o n of c a f f e i n e from the Chinese gorgonian Echinogorgia pseudosappo can perhaps be r a t i o n a l i z e d by the c h a r a c t e r i s t i c a b i l i t y of gorgonians to incorporate plant c e l l s i n a symbiotic fashion into t h e i r t i s s u e s . The occurrence of doridosine (59) i n both a sponge and a nudibranch dig e s t i v e system indicates that the compound o r i g i n a t e d i n the sponge which was grazed upon by the nudibranch. A case could be made for the sponge obtaining the purine d e r i v a t i v e from a symbiotic d i e t a r y micro-organism. Sponges feed on b a c t e r i a and they contain many symbiotic microalgae. Observation of ageline A (66) and hokupurine (73) i n more than one organism i s further proof of the d i f f i c u l t y encountered i n the assignment of o r i g i n f o r these purine metabolites. Nitro containing metabolites are very rare i n nature, e s p e c i a l l y from marine sources. Fungi have been reported to produce a few such compounds. One of the f i r s t reported n i t r o containing compounds was /3-nitropropionic a c i d (74), i s o l a t e d i n 1951 by Bush et a l . 5 ^ from A s p e r i g i l l u s flavus. This compound was found to have a very low l e v e l of a n t i b i o t i c a c t i v i t y . Two new oxindole a l k a l o i d s , cyclopiamine A (75), and i t s enantiomer, cyclopiamine B (76) have been i s o l a t e d from - 61 -P e n i c i l l i u m cvcloplum.^3 P. cyclopium Westling i s a frequently encountered fungus on stored grain and cereal products destined f or human or animal consumption. 76 T h a l l e r at a l . ^ 4 i n 1972 reported i s o l a t i o n of p_-nitrobenzaldehyde (77) from the fungus Lepista diemii Singer. The f i r s t example of a n i t r o containing phenol appears to be 3,5-dinitroguaiacol (78) reported by Ohta et al.^5 i n 1977 from the red 62 alga N a r g i n i s p o r a m a b e r r a n s. To the best of this authors knowledge, the isolation of nitrophenols by Ayer et a l . 2 9 i n 1984 was only the second example from the marine environment. It is interesting to note that the low yield of 3,5-dinitroguaiacol (78), 10 mg from 15 Kg of alga, also suggests the possibility of a microbial origin. In conclusion, the compounds isolated from the Northeast Pacific bryozoans seem to be of a dietary or symbiotic origin. The dietary origin of bryozoan metabolites has been suggested by Pettit et al.^^a ^ n the isolation of the bryostatins from two bryozoans, a tunicate and a sponge. The bryostatins appear to be biogenetically related to a number of dinoflagellate toxins such as the pectenotoxins. Similarly, nitrophenols 38 and 39 have recently been isolated by Northcote,^6b f r o m a tunicate, Halocynthia i i abopa. and a sponge, Leucosolenia sp., collected in Barkley Sound, B.C. lending further credence to the hypothesis of dietary or symbiotic origin of these compounds. Conforma-tion of this hypothesis could only come through the isolation of the family of nitrophenols from some micro-organism, whether i t be phyto-planktonic, bacterial or fungal in nature. - 63 -E. INTRODUCTION TO THE SPONGES Sponges (Phylum Porifera) are the most primitive of multicellular animals. They contain a relatively simple internal organisation, lacking true tissue and organs. A l l members of the phylum are sessile and exhibit l i t t l e detectable movement. Sponges are primarily marine animals, except for around 150 fresh water species. They are found in a l l seas wherever there are rocks, shells, submerged timbers or coral to provide a suitable substratum for attachment. Sponges vary greatly in size. Certain calcareous species are about the size of a bean, while others can reach the size of a square meter. Some species are radially symmetrical while the majority exhibit irregu-lar, massive, erect, encrusting or branching growth patterns. The size and shape are generally influenced by the nature of the substratum, ava i l a b i l i t y of space, and velocity and type of water current. The water current brings in oxygen and food and removes waste. Remarkably, the volume of water estimated to pass through a sponge 10 cm in height and 1 cm in diameter is about 27.5 L per day. Marine sponges feed on extremely small particles. It has been demonstrated that the majority of the matter consumed by sponges is of a size undetectable by an ordinary microscope, while the other food consumed consists chiefly of bacteria, dinoflagellates and other plank-ton. Sponges reproduce asexually by budding or by a variety of pro-cesses which involve formation and release of an aggregate of essential cells from the parent sponge. In marine sponges, these aggregates are - 64 -c a l l e d gemmules. Formation of these gemmules i n great numbers takes place i n the f a l l before the parent sponge disint e g r a t e s with the onset of winter. These gemmules are able to withstand f r e e z i n g and drying enabling the species to e x i s t through winter. In the spring, the i n t e r i o r c e l l s of the gemmules emerge u l t i m a t e l y developing into an adult sponge. The approximately 10,000 species of known sponges can be placed w i t h i n four classes based on the nature of the skeleton. Class Calcarea, comprises a l l members known as calcreous sponges, distinguished by spicules composed of calcium carbonate. Sponge sp i c u l e s , made up of calcium carbonate or s i l i c o n dioxide deposits, vary i n s i z e and shape and often serve as useful characters i n i d e n t i f y i n g sponges. Spicules are generally l a b e l l e d by the number of axes or rays they possess by adding the appropriate numerical p r e f i x to the ending -axons (when r e f e r r i n g to the number of axes) or -actine (when r e f e r r i n g to the number of rays or p o i n t s ) . The spicules of calcarea are monaxins or three or four pronged types, u s u a l l y separate. The colours usually encountered i n calcareous sponges vary from greyish white to b r i l l i a n t yellow, red or lavender. Species of t h i s c l a s s are the smallest of a l l sponges ( l e s s than 10 cm i n height). They can be found i n a l l the oceans of the world, but are generally r e s t r i c t e d to shallow waters. Class H e x a c t i n e l l i d a , commonly known as "glass sponges", get t h e i r name from the f a c t that the spicules are always of the t r i a x o n or s i x pointed type. Also, some of the spicules are o c c a s i o n a l l y fused to form a l a t t i c e l i k e skeleton b u i l t of long s i l i c e o u s f i b e r s , hence t h e i r common name. This c l a s s elaborates the most symmetrical sponges, which 65 -have cup, vase or urnlike shapes averaging 10 to 30 cm in height. In contrast to Calcarea, Hexactinellidae are mainly deep water sponges, found at depths of 400 to 950 meters mainly in tropical waters of the West Indies and the Eastern Pacific from Japan to the East Indies. Class Demospongiae contains the greatest number of species includ-ing most of the North American sponges. The majority are marine and distributed from shallow water to great depths. Different species are characterized by different bright colours due to pigment granules in their c e l l s . The skeletons vary, consisting of siliceous spicules or spongian fibers or a combination of both. Spicules containing species differ from those in Class Hexactinellida in that their spicules are larger manoxins or tetraxons rather than triaxons. Finally, Class Sclerospongiae sponges diff e r from other sponges in having an internal skeleton of siliceous spicules and spongian fibers and an outer encasement of calcium carbonate. Sponges, particularly those without spicules, often produce large quantities of interesting and biologically active secondary metabolites that are thought to deter potential predators and inhibit growth of fouling organisms. Since many sponges contain symbiotic micro-organisms, the true origin of these compounds is at times in question, however, the majority have been attributed to the sponges. As evidence for the use of these compounds for self defence, i t has been observed that in the sponge Aplysina f i s t u r l a r i s secondary metabolites can only be found in the cells localized adjacent to the exhalent canals. 5 7 Pioneering studies by Bergmann5^ on the chemical and ecological aspects of marine sponges in the early 1960's initiated intense studies - 66 -leading to the i s o l a t i o n of hundreds of s t r u c t u r a l l y v a r i e d natural products e s p e c i a l l y s t e r o l s , many of which were common to t e r r e s t r i a l sources plus a few unique to the marine environment. Due to a lack of s e n s i t i v e a n a l y t i c a l techniques i n t h i s period, i n v e s t i g a t o r s were l i m i t e d to the study of only the major components, therefore neglecting the often more i n t e r e s t i n g trace and minor metabolites. Studies dating back to the 1940's and 1960's, therefore, y i e l d e d mainly "conventional" s t e r o l s , that i s , s t e r o l s which possess the normal 19 carbon nucleus plus an 8 to 10 carbon side chain. Among the most common s t e r o l s found i n sponges at t h i s time were c l i o n a s t e r o l (79) and p o r i f e r a s t e r o l (80) i s o l a t e d o r i g i n a l l y from Cliona celata.-* 9 Another 29 carbon s t e r o l , c h o n d r i l l o s t e r o l (81), reported as a major component 81 - 67 -of C h o n d r i l l a n u c u l a . ^ seemed at the time to be s o l e l y confined to sponges. Among the most widely d i s t r i b u t e d 27 and 28 carbon s t e r o l s i n marine organisms are 24-methylenecholesterol (82), found o r i g i n a l l y i n Chalina arbuscula.^ 1 c h o l e s t e r o l (83) and cholestanol (84) found as major components i n numerous sponges. 84 With the advent of increased a n a l y t i c a l technology i n the early 1970's the r e i n v e s t i g a t i o n of marine sponges has r e s u l t e d i n the i s o l a -t i o n and documentation of s t e r o l s possessing completely unprecedented side chain a l k y l a t i o n patterns and modified t e t r a c y c l i c n u c l e i . In the - 68 -1970's to 1980's reports of unusual s t e r o l s began to appear i n the l i t e r a t u r e . Remarkable new side chains including those containing cyclopropane and cyclopropene units, i n addi t i o n to extensively alky-l a t e d side chains were reported. In 1975, Fattorusso et al.^2 reported i s o l a t i o n of c a l y s t e r o l (85) from the sponge, Calyx niceaensis. C a l y s t e r o l (85) possessed a cyclopropenyl u n i t i n the side chain, a feature previously observed i n nature only i n some f a t t y acids. 85 L i et a l . 0 J l a t e r reported another two s t e r o i d a l cyclo-propenes, (23R),23H-isocalysterol (86) and (24S), 24H-isocalysterol (87) from Calyx niceaensis. along with two s t e r o i d a l cyclopropane deriva-t i v e s , (23R,24R,28S),23,24-dIhydrocalysterol (88) and (23S,24S,28R)-23,24-dihydrocalysterol (89). A novel cyclopropane containing s t e r o l , n i c a s t e r o l (90), was reported i n 1985 by Proudfoot et a l . 6 4 from Calyx  niceaensis. The proposed structure was confirmed by p a r t i a l synthesis. Many s t e r o l s with "extended" side chains have been encountered i n sponges. These include a p l y s t e r o l ( 9 1 ) , s t e l l i f e r a s t e r o l (92) and - 69 -i s o s t e l l i f e r a s t e r o l (93), which a l l are characterized by the unusual a d d i t i o n of an extra carbon at C-26. Three further examples of extended side chains are found i n veron-g u l a s t e r o l (94), a minor s t e r o l from the sponge Verongia cauliformis.^6 and i n x e s t o s t e r o l (95), a major component, and xestostanol (96), a 70 minor component of Xestosponpia muta. ' These three sterols possess the R unique feature of being alkylated at both the C-26 and C-27 positions. Recent isotope feeding studies have led to the demonstration of de novo biosynthesis of xestosterol (95) from desmosterol (97), via a nonstereoselective SAM biomethylation and a 1,2 hydrogen shift (Scheme 20). 6 8 Modifications to the sterol nucleus are also well documented from sponges. A mixture of stanols having a 19-norcholestanol nucleus carrying either saturated or unsaturated 8, 9 or 10 carbon side chains has been isolated from the sponge Axinella polypoides.^9 The major component, ful l y characterized by spectral data as 19-nor-5a,10/?-ergost-trans-22-en-3;9-ol (98), has been found along with related structures (99)-(105). Scheme 20: Biosynthesis of xestosterol (95) 9 9R - H -|04 R - Et, A 2 2-trans 105 100R - Me 72 -E x a m i n a t i o n o f t he sponge A x i n e l l a v e r r u c o s a 7 0 i n 1974 l e d t o the i s o l a t i o n o f t he f i r s t example o f an A - n o r c h o l e s t a n e n u c l e u s f rom the m a r i n e e n v i r o n m e n t . As many as s i x 3 / ? - h y d r o x y m e t h y l - A - n o r - 5 a - c h o l e s t a n e s t e r o l s , ( 1 0 6 ) - ( 1 1 1 ) , c o n t a i n i n g v a r i o u s s i d e c h a i n s , were i s o l a t e d . H 0 H 2 C H 106 R - H 107 R - Me 108 R - Et 109 R - H, A 2 2 110 R - Me, A 2 2 111 R - Et, A 2 2 I n 1982 , E g g e r s d o r f e r e t a l . 7 1 r e p o r t e d the i s o l a t i o n o f 3 /S -hydroxy-m e t h y l - A - n o r - 5 a - c h o l e s t - 1 5 - e n e (112) f rom t h e p a c i f i c sponge , 112 - 73 -Homaxinella trachys. This represents the f i r s t n a t u r a l l y occurring s t e r o l with a C-15, C-16 double bond. Biosynthetic tracer incorporation studies c a r r i e d out by Bibolino et a l . ' ^ suggest the A-nor s t e r o l s can be formed de novo from r e a d i l y available c h o l e s t e r o l v i a an enzyme catalyzed r i n g contraction mechanism invol v i n g the loss of the 3a and 4/3 hydrogens and the formation of a C-C linkage between C-2 and C-4 (Scheme 21). Scheme 21: Biosynthetic conversion of cholesterol into 3/3-hydroxy-methyl-A-nor-cholestane - 74 This is the most convincing evidence so far for the abi l i t y of sponges to chemically modify sterols subsequent to dietary intake. It has also been found that sponges which contain these A-nor sterols lack 7 ^  sterols with conventional nuclei. This possibly indicates the efficiency of the enzymic system in converting absorbed dietary sterols into A-nor sterols. Therefore, the existence of as yet unknown sterols in nature with conventional nuclei but unusual side chains can be detected in some cases through isolation of the corresponding A-nor sterol. The most common theories on the diversity of marine sterols appear to be due to the accumulation of several phenomena, including genetic control, biosynthesis by zooxanthellae, commensalism and parasitism, and transmission of molecules by the food chain or by sea water. From a biochemical standpoint, structural variation in the form of extended side chains via biological methylations has been shown to modulate c e l l p l a sticity, membrane permeability and ion exchange. 7 4 The effect of nuclear modification of sterols is as yet unclear, however, i t is believed to also play a specialized role in membrane function. - 75 -F. NOVEL STEROIDS FROM THE SPONGE Anthoarcuata graceae (Bakus 1966) 1. Introduction Anthoarcuata graceae (Bakus 1966) is a reddish orange sponge (family Plocamiidae, order Poccilosclida, class Demospongiae, subclass Homoscleromorpha) commonly found in rocky intertidal and subtidal habitats of the B.C. coast. The dorid nudibranch Aldisa sanguinea  cooperi is often found in close association with the sponge.7-5 Most often the nudibranch is deeply embedded in the sponge A^ graceae from which i t obtains nutrition and pigments giving i t cryptic coloration. Our chemical studies on Aj. graceae were prompted by a prior investigation of the chemistry of the nudibranch A^ . cooperi which yielded a mixture of biologically active steroids 113 and 114, a glycerol ether 115 and a complex mixture of minor steroidal ketones, fats and sterols which were not studied further. 7-* A. graceae. collected in the shallow waters of Barkley Sound, B.C. at various times of the year was found to be lacking in the major metabolites found in A^ . cooperi. however, a preliminary investigation did reveal the presence of several other minor but very interesting steroids. - 76 -115 2. Isolation and Structure Elucidation Anthoarcuata praceae was collected by hand using SCUBA (1-5 m depth) and immediately immersed in methanol (or ethanol). After extraction for one to three days at room temperature, the methanol (or ethanol) was decanted, vacuum f i l t e r e d and evaporated in vacuo to yield an aqueous methanolic suspension. This suspension was partitioned between brine and chloroform, and the organic layer was dried over anhydrous Na2S0^. The sponge was further soaked in dichloromethane at room temperature for one day, before being ground (with dichloromethane) in a Waring blender. The suspension of ground sponge in dichloromethane was vacuum f i l t e r e d through celite to yield a f i l t r a t e which was stored - 77 -at below room temperature. The resulting sponge solids were extracted with dichloromethane a further three times at one day intervals. The combined f i l t r a t e s were evaporated in vacuo, partitioned between brine and chloroform, and the organic layer was dried over anhydrous N a 2 S 0 ^ . F i l t r a t i o n and evaporation in vacuo of the combined organic layers afforded a dark red crude o i l which was fractionated by flash chromato-graphy to give complex mixtures of fats, steroids and pigments detected by analytical TLC analysis. Further purification of the steroidal fraction by column chromatography (Sephadex LH -20) and reverse phase HPLC yielded two steroidal fractions as white solids. Analysis of these two fractions by H^ and 1 3C NMR provided evidence for the presence of a mixture of components in each. Further separation and purification of each mixture was carried out by preparative TLC yielding two novel A 4-3,6-diketosteroids 116 and 117, two unprecedented A-nor-steroids anthosterone A (118) and anthosterone B (119) as well as two diosphenols tentatively assigned structures 120 and 121. 3A. A 4-3 ,6-diketosteroids Compound A, mp 92-93°C, had a molecular formula of C 2 7 H 4 0 O 2 (HRMS 396.3034, calcd. 396.3030) that required 8 units of unsaturation. The *H NMR indicated methyl resonances at 6 0 . 7 3 (s, 3H) , 0 . 9 5 (d, J - 6 Hz, 3 H ), and 1.17 ppm (s, 3 H ), corresponding to the C-18, C - 2 1 , and C-19 methyls of a conventional steroid. In addition, two olefinic methyl H^ NMR resonances were found at 6 1.61 (s, 3 H ) , 1.69 (s, 3 H ), as well as an 78 -120 - 79 -olefinic t r i p l e t at 5.09 (t, J - 6.4 Hz, IH), ppm, which indicated the presence of a A 2 4 • 2 5 double bond in the side chain. This was further supported by an IR absorbance at 1643 cm"1 and 1 3C NMR resonances at 6 124.99 (d), 127.09 (s) ppm which could be assigned to the C-24 and C-25 carbons of the side chain. One remaining olefinic signal in the ^ H NMR at S 6.19 (s, IH) ppm (Figure 11), in addition to four downfield resonances in the 1 3C NMR, indicated the presence of some unique functionality in the A and B rings. The 1 3C NMR resonances at 5 202.36 (s) and 199.56 (s) ppm were reminiscent of the carbonyl carbon resonances found in A 4-3-ketosteroids (Table 8), while the remaining two resonances at 8 125.48 (d) and 161.06 (s) ppm could be assigned to the corresponding A4-double bond. Infrared bands at 1606 (vc - c) amd 1686 (i/c - ojcm"1, coupled with a UV A m a x at 248.9 nm (e 11600) representing an enone with extended conjugation, allowed the tentative assignment of compound A 116 as an ene-dione. Confirmation of the assigned structure was achieved by the synthe-sis of a model A 4-3,6-diketosteroid system (Scheme 22). This was Scheme 22: Jones oxidation of c h o l e s t e r o l (83) Figure 12: 400 MHz XH NMR of compound 122 - 83 -Table 7: 1 H NMR Data for A H-3,6-Ketosteroids (CDCI3) Chemical Shift, 5, ppm H on C # (116) a (117) a (122) a 4 6 19 (s,lH) 6 17 (s.lH) 6.19 (s, IH) 18 73 (s,3H) 73 (s,3H) .74 (s, 3H) 19 1 17 (s,3H) 1 17 (s,3H) 1.19 (s, 3H) 21 95 (d,J=6 Hz,3H) 85 (d,J=7 Hz,3H) .94 (d, J=6 Hz, 3H) 24 5 09 (t,J°6.4 Hz.lH) - -26 1 69 (s,3H) b 1 02 (d,J=8 Hz,3H) .87 (d, J=6 Hz, 3H) 27 1 .61 (s,3H) b - -28 4 .65/4.73 -400 MHz shifts interchangeable 116 11 7 0 Figure 14: 400 MHz iH NMR of compound 117 - 86 -Table 8: i JC NMR Data for A 4 -3,6-diketosteroids ( C D C I 3 ) Chemical Shift, 6, ppm H on C # (116) (117) (122) 3 199. ,56 (s) 199. ,56 (s) 199. ,40 (s) , 4 125. .48 (d) 125, .44 (d) 125. .33 (d) 5 161. ,06 (s) 161. ,08 (s) 160, ,98 (s) 6 202, .36 (s) 202. ,36 (s) 202. ,25 (s) 24 124. ,99 (d) 156. .65 (s) 25 127, ,09 (s) 28 - 106, .06 (t) 1 2 2 - 87 -Scheme 23: MS fragmentation of compound B 117 - 88 -achieved via a Jones oxidation 7^ of cholesterol (83) which after purification by preparative TLC gave A4-cholestane-3,6-dione (122) as one of the major products. The HRMS of 122 gave a parent ion corres-ponding to a molecular formula of C27H42^2 (HRMS 398.3186, calcd. 398.3187). A 1H NMR spectrum of 122 (Table 7) showed a sharp olefinic signal at 5 6.19 (s, IH) ppm (Figure 12) and its 1 3C NMR spectrum (Figure 13) had resonances at S 199.40 (s) and 202.25 (s) corresponding to the carbonyl carbons (C-3, C-6) plus two olefinic resonances at 6 125.33 (d) and 160.98 (s) ppm (C-4, C-5) assigned to the A 4• 5 double bond system (Table 7). The IR spectrum of 122 showed absorbances at 1685 and 1606 cm"1 in close agreement to the natural product. Co-occurring with Compound A 116 was a related steroid, Compound B 117, mp 111-112°C, which possessed a A 2 4 , 2** double bond in the side chain. Compound B 117 had a molecular formula C 2 8 H 4 2 O 2 (HRMS 410.3190, calcd. 410.3187) differing from compound A 116 by 14 mass units ( = C H 2 ) . The lH NMR (Figure 14) showed methyl resonances at 6 0.73 (s, 3H), 0.85 (d, J = 7 Hz, 3H), 1.02 (d, J - 8 Hz, 6H), 1.17 (s, 3H) ppm correspond-ing to the C-18, C-21, C-26/27 and C-19 methyls respectively, of the steroidal skeleton (Table 7). H^ NMR olefinic resonances at 6 4.65 (s, IH) and 4.73 (s, IH) ppm, in addition to 1 3C NMR (Figure 15) signals at S 106.06 (t) and 156.65 (s) ppm, are typical of an olefinic-methylene at the C-24/28 position (Table 8). This is further supported by the strong peak at m/z 326 in the mass spectrum indicating a loss of the ^6^12 s l c*e chain via a McLafferty rearrangement (Scheme 23) . Identical functionality in the A and B rings was confirmed by the presence of a sharp olefinic singlet at 5 6.17 (s, IH) ppm in the H^ NMR, and by - 89 -resonances at S 125.44 (d), 161.08 (s) (A 4' 5-double bond), 199.56 (s), 202.36 (s) ppm (carbonyl carbons) in the 1 3C NMR. Compounds A and B had comparable UV and IR spectral data. 3B. A-Nor Steroids Examination of the more polar band from the preparative TLC puri-fication yielded two related compounds anthosterone A (118) and anthos-terone B (119). Anthosterone A (118), mp 142-143°C, had a molecular formula C28H42°4 (HRMS 442.3083, calcd. 442.3079) requiring 8 units of unsaturation. Its NMR spectrum gave methyl signals at 8 0.73 (s, 3H), 0.96 (d, J = 6.7 Hz, 3H), and 1.21 (s, 3H) ppm corresponding to the C-18, C-21 and C-19 methyls of a steroid skeleton, in addition to two signals assigned to olefinic methyl groups (C-26/C27) at 8 1.62 (s, 3H), and 1.69 (s, 3H) ppm. Further examination of the ^H NMR spectrum (Figure 16) indicated the presence of an olefinic t r i p l e t at 8 5.10 (t, J - 8 Hz, IH) ppm which could be assigned to a proton on C-24 of a A24,25 double bond in the side chain (Table 9). 1 3C NMR olefinic resonances at 125.09 (d) and 131.05 (s) supported this assignment (Table 10). Additional H^ NMR resonances consisted of an AB quartet made up of doublets resonating at 8 2.08 (d, J - 13.3 Hz, IH) and 2.19 (d, J - 13.3 Hz, IH), one sharp methyl singlet at 3.79 (s, 3H), and a deshielded olefinic proton at 6.73 (t, J = 3.3 Hz, IH) ppm. 90 -- 91 -92 -- 93a--93 b -94 -Table 9: •LH NMR data f o r compounds 118 and 119 Chemical s h i f t , 6 ppm H on C # 118 a 119 a 1 2.08/2.19 (d, J-13.3 Hz, IH) 2.10/2.18 (d, J-13.5 Hz, IH) 5 6 73 ( t , J=3.3 Hz, IH) 6.73 ( t , J=2 5 Hz, IH) 18 0 73 (s, 3H) 0.73 (s, 3H) 19 1 21 (s, 3H) 1.20 (s, 3H) 21 0 96 (d, J=6.7 Hz, 3H) 0.97 (d, J=6 5 Hz, 3H) 24 5 10 ( t , J=8 Hz, IH) -26 1 62 b (s . 3H) 1.04 (d, J-6 5 Hz, 6H) 27 1 69 b (s , 3H) 28 3 79 (s, 3H) 4.65-4.71 (s IH) 29 3.77 (s, 3H) 11 8 119 - 95 -Table 10: i JC NMR data for compounds 118 and 119 Chemical shift, 5 ppm H on C # 1181 H9T a 1 56 02 (t) 46 68 (t) 2 79 90 (s) 79 89 (s) 3 173 37 (s) 173 36 (s) 4 200 91 (s) 200 91 (s) 5 145 10 (s) 145 08 (s) 6 136 28 (d) 136 29 (d) 7 32 21 (t) 32 21 (t) 8 32 32 (d) 32 32 (d) 9 49 66 (d) 49 64 (d) 10 42 94 (s) 42 95 (s) 11 22 11 (t) 22 01 (t) 12 39 92 (t) 39 93 (t) 13 39 35 (s) 39 34 (s) 14 56 26 (d) 56 25 (d) 15 24 35 (t) 24 34 (t) 16 28 11 (t) 28 13 (t) 17 55 91 (d) 55 93 (d) 18 12 03 (q) 12 03 (q) 19 21 79 (q) 22 11 (q) 20 35 55 (d) 35 72 (d) 21 18 66 (q) 18 74 (q) v 22 24 35 (t) 30 99 23 36 06 (t) 34 64 ( t ) b 24 125 09 (d) 153 53 (s) 25 131 05 (s) 33 81 (s) 26 17 66 (q) 21 87 (q) c 27 25 75 (q) 21 79 (q) c 28 53 39 (q) 106 01 (t) 29 53. 40 (q) t Assignment based on APT experiment (Figure 20). a 75 MHz, CDC13 k Assignments interchangeable c Assignment interchangeable 96 Assignment of the sharp methyl singlet at 5 3.79 was aided by the observation of a MS fragment at m/z 383.2932 (calcd. 383.2952) indicat-ing an M+-59 loss corresponding to C2H3O2 of a methyl ester function-a l i t y . The presence of the methyl ester moiety was further confirmed by 1 3 C NMR resonances at 6 173.37 (s) (C-4, C-0) and 55.91 (q) (C-29, CH3) ppm (Figure 17) along with an IR band at 1744 cm"1. An interesting absorbance in the IR spectrum at 1717 cm"1, coupled with a 1 3C NMR carbonyl signal at 6 200.91 (s), two olefinic signals at 6 136.28 (d) and 145.10 (s) ppm, and a UV A m a x at 250.2 (c 12500) nm suggested the presence of an enone system as seen in compounds A and B. The mass spectrum of 118 also showed a strong peak at m/z 330.1810 (C20H26°4> calcd. 330.1832) indicating a M+-113 (C gH 1 7) loss correspond-ing to the cleavage of the side chain (Scheme 24). With the further loss of 60 daltons (C2H4O2) as indicated by a peak at m/z 270.1597 (calcd. 270.1621), the remaining nucleus after the loss of the methyl ester yielded a molecular formula C^gH220 2 which f i r s t indicated the presence of a nor-sterol (Scheme 24). With a l l the methyl groups accounted for, a logical conclusion to draw was the presence of an A-nor ring. Co-occurring with anthosterone A (118) was the related steroid, anthosterone B (119), mp 155-157°C, which had a molecular formula C29 H44°4 (HRMS 456.3236, calcd. 456.3241) requiring 8 units of unsatu-ration. The 1H NMR (Figure 18) showed methyl signals at 6 0.73 (s, 3H), 0.97 (d, J = 6.5 Hz, 3H), 1.04 (d, J = 6.5 Hz, 6H), 1.20 (s, 3H) ppm assigned to the C-18, C-21, C-26/27 and C-19 methyls respectively. A difference in molecular weight of 14 daltons (CH2) plus olefinic - 97 -Scheme 24: MS fragmentation of anthosterone A (118) m/z 383 (10 V.) resonances in the 1H NMR at 6 4.65 (s, IH), and 4.71 (s, IH) ppm indicated the presence of a C-24 (28) olefinic-methylene system (Table 9). This assignment was supported by the 1 3C NMR resonances at 106.01 (t) and 156.79 (s) ppm (Figure 19) which are diagnostic for this type of system. The remaining portions of the ^ H NMR spectrum including the presence of the AB quartet (2.10/2.18, d, J - 13.3 Hz, 2H), a sharp singlet at 6 3.77 (s, 3H) for the methyl ester as well as an olefinic t r i p l e t at 6.73 (t, J - 3 Hz, IH) ppm indicated that the basic nucleus - 98 -of anthosterones A and B was identical. This was supported by the ^-^C NMR spectrum which showed a deshielded carbonyl resonance at <5 200.91 (s), two olefinic resonances at 136.29 (d) and 146.08 (s) ppm (Table 10) and the mass spectrum which also showed a strong peak at 298.1928 (calcd. 298.1934) corresponding to the C20H26O2 nucleus (Scheme 25). Scheme 25: MS fragmentation of anthosterone B (119) 119 m/z 456 ( 2 •/•) m/z 382(29'/.) m/z 298 (48 V.) Since the difference between the two compounds was only in their side chains, the remaining objective was to propose a structure for t h e common nucleus. Further examination of the NMR spectrum of anthosterone A (118) showed the presence of a carbon singlet at 6 79.90 (s) assigned t o a - 99 -tertiary carbinol carbon. The presence of a hydroxyl group was sup-ported by the mass spectral loss of HOH (Scheme 24). As expected then, anthosterone B (119) could be acetylated with acetic anhydride/pyridine at room temperature for 12 hr to afford anthosterone B-acetate (123) 498.3336 (calcd. 498.3347) in quantitative yield (Scheme 26). 7 7 Scheme 26: Acetylation of anthosterone B (119) 123 The !H NMR spectrum of 123 (Figure 21) showed an upfield shift in the C-19 methyl resonance from 5 1.20 in 119 to 1.10 ppm in 123, as well as a downfield shift in both protons making up the AB system, with the doublets at 5 2.10 and 2.18 in 119 shifting to 2.47 and 2.65 ppm respectively in 123 (Table 11). This indicated a close proximity in the - 100 -101 Table 11: *H NMR comparison between 119 and 123 0O 123 H on C # Chemical shift, 5 ppm 119' 123' 18 19 21 26 27 2.10 (d, J-13.5 Hz, IH) 2.18 (d, J=13.5 Hz, IH) 0.73 (s, 3H) 1.20 (s, 3H) 0.97 (d, J-6.5Hz, 3H) 1.04 2.47 (d, J=14 Hz, IH) 2.65 (d, J - 15 Hz, IH) 0.72 (s, 3H) 1.10 (s, 3H) 0.97 (d, J=8 Hz, 3H) 1.03 (d, J-8 Hz, 6H) 28 4.65/4.71 (s, IH) 4.67-4.73 (s, IH) - 1 0 2 -nucleus between the hydroxyl group, the C-19 methyl and the AB spin system. A difference NOE experiment, in which the C-19 methyl singlet of anthosterone B (119) was irradiated, showed an enhancement of the upfield portion of the AB quartet showing the close spacial proximity of these functionalities. Additional NOE experiments included irradiation of the olefinic t r i p l e t at 6" 6.73 ppm which induced an enhancement of a l l y l i c proton multiplets at 6 1.88 (m, IH), and 2.41 (m, IH) ppm and irradiation of the multiplet at 6 2.41 (m, IH) yielding an NOE enhance-ment of the t r i p l e t at S 6.73 as well as i t s apparent geminal partner at 1.88 (m, IH) ppm. Irradiation of the olefinic signal at 6 6.73 in a double resonance experiment resulted in sharpening of signals at 1.88 and 2.40 ppm, while irradiation of the multiplet at S 2 AO changed the tr i p l e t at 6.73 to a doublet and sharpened the signal at 1.88 ppm. The 1 3C NMR spectrum showed two olefinic signals, 6 136.28 (d) and 145.10 (s) ppm, which could be assigned to a t r i - substituted alkene with an adjacent a l l y l i c methylene (Figure 22). Figure 22: A l l y l i c methylene system - 103 -The next major obstacle was the positioning of the ketone carbonyl which from the UV data had to be conjugated to a double bond. Assuming the presence of the contracted A ring, i t was possible to propose structures based on a l l the accumulated data which possessed either a 4-keto or a 6-keto functionality. Three possible structures A, B, and C were proposed which accommodated a l l the functionality required by the data, including the methyl ester, enone system, tertiary alcohol as well as the five membered A ring. G A SINEPT7^ (selected insensitive nuclei enhanced by polarization transfer) experiment eliminated one of the po s s i b i l i t i e s based on observed polarization transfer through 2 and 3 bond - 1 3C couplings. A soft pulse applied to the upfield proton of the AB system (5 2.18 ppm) afforded enhancements of 1 3C NMR signals at 200.91 (s), 173.36 (s), - 104 -145.08 (s) and 79.90 (s) ppm indicating -^H - 1 3C coupling to the a,/3 unsaturated ketone carbonyl, the methyl ester carbonyl, the non-protonated olefinic and the carbinol carbons (Figure 23). Only struc-tures A and C could f i t this data while with structure B, a required 6-bond coupling to the methyl ester carbonyl carbon would not be expected. A two dimensional homonuclear COSY79 experiment confirmed that the AB system was not spin isolated as indicated by an observed coupling between the downfield member of the AB system at 6 2.18 and the C-19 methyl singlet at 1.20 ppm (Figure 24). This observed coupling (W-coup-ling) between the methyl group and the downfield a-proton provided strong evidence for the proposed structure C. The relative stereochem-istry at the C-2 center with the hydroxyl group in the 0 orientation, was assigned based on the observed upfield shift of the C-19 methyl resonance (1.20 to 1.10 ppm) on acetylation of anthosterone B (119). Confirmation of the structure was provided by a single crystal X-ray diffraction a n a l y s i s 8 0 on anthosterone A (118). Figure 25 shows a computer generated ORTEP drawing of 118. IRRAD. AT 2.18 ppm 200 .91 Figure 23: SINEPT experiment on anthosterone B 119 - 106 -Figure 24: 2D Homonuclear COSY on anthosterone B (119) 2^ ( P " V ) 0 4 -| 3 . 2 2 8 2 . 4 2 0 1 .6 1 .2 0 . 6 0 4 F l (PPM) Figure 25: Computer generated ORTEP drawing of anthosterone A 118 - 107 -3C. Diosphenols 120 and 121 An investigation of two steroids which were more polar than the A-nor sterols on preparative TLC, and present in trace amounts, has led to a preliminary structural proposal based on spectral data. Compound E 120, had a molecular formula C27H42O3 (HRMS 414.3149, calcd 414.3136) requiring 7 units of unsaturation. The NMR spectrum gave methyl signals at S 0.73 (s, 3H), 0.93 (d, J -= 6 Hz, 3H), and 1.28 (s, 3H) ppm assigned to the C-18, C-21 and C-19 methyls of a steroid nucleus. Two olefinic methyl groups (C26/27) at 6 1.52 (s, 3H) and 1.69 (s, 3H) plus an olefinic t r i p l e t at 5.09 (t, J - 7 Hz, IH) ppm indicated the presence of a trisubstituted side chain double bond as seen in compound A 116 and anthosterone A (118). Further examination of the H^ NMR (Figure 26) revealed an AB system, 6 2.18 (d, J = 16 Hz, IH) and 2.25 (d, J = 16 Hz, IH), a sharp singlet at 3.29 (s, 3H), a deshielded methine proton at Figure 26: 400 MHz LH NMR of compound E 120 - 109 4.52 (t, J =3 Hz, IH) as well as an olefinic singlet at 5.28 (s, IH) ppm (Table 12). Based on a loss in the mass spectrum of 32 daltons (MeOH), the three proton singlet at 3.29 was assigned to a methyl ether functionality (Scheme 27). Infrared bands at 3438 (i>0-H) , 1711, 1674 (wC=0) and 1606 (i/C=C) cm"1, plus a UV absorbance 264 nm (e 10971) which shifted to 304 nm (e 9731) on addition of base, strongly indicated the presence of a dios-phenol functionality based on a comparison with literature values.^ The presence of the diosphenol was further supported by the positive TLC spray test with 2,4-dinitrophenylhydrazine^ 2 and FeC^.^ 3 110 Table 12: XH NMR data on diosphenols 120 and 121 Chemical s h i f t , 6 ppm H on C # 120 a 121 a 1 2.18/2.25 (d, J-16 Hz, IH) 2.17/2. 29 (d, J-16 Hz, IH) 4 5.28 (s, IH) 5.28 (s, IH) 6 4.52 ( t , J-3 Hz, IH) 4.57 (t , J-2 Hz, IH) 18 0.73 (s, 3H) 0.74 (s. 3H) 19 1.28 (s, 3H) 1.31 (s, 3H) 21 0.93 (d, J - 6 Hz, 3H) 0.94 (d, J - 8 Hz, 3H) 23 5.09 (s, IH) -25 1.52 b (s, 3H) 1 \ 1.03 (d, J - 7 Hz, 6H) 26 1.69 b (s, 3H) J 27 3.29 4.76 (s, IH) 28 - 3.33 (s, 3H) 400 MHz, CDC1 3 s h i f t s interchangeable OCH3 OCH3 120 121 - I l l -Irradiation of the C-19 methyl singlet at 6 1.28 in a difference NOE experiment on compound E 120, resulted in an enhancement of the methoxy singlet at 3.29 and the upfield component of the AB system at 2.18 (d, J = 16 Hz, IH) ppm. Irradiation of the t r i p l e t at 5 4.52 ppm afforded only an enhancement of the methoxy singlet. Decoupling of the tr i p l e t at 8 4.52 sharpened a l l y l i c signals at 2.10 as well as proton signals in the aliphatic region at 1.00 to 1.20 ppm. A two dimensional homonuclear COSY experiment on compound E further suggested coupling between the proton at <S 4.52 and an a l l y l i c proton at 2.10 and aliphatic protons at 1.00 to 1.20 ppm (Figure 27). Co-occurring with compound E 120 was the related compound F 121, which had a molecular formula of C28H44O3 (HRMS 428.3284, calcd. 428.3291). A 1H NMR of compound F 121 afforded signals at 6 0.74 (s, 3H), 0.94 (d, J - 7 Hz, 3H), 1.03 (d, J - 6.4 Hz, 3H), and 1.31 (s, 3H) ppm assigned to the C-18, C-21, C-26/27 and C-19 methyls respectively. The presence in the ^ H NMR (Figure 28) of sharp olefinic singlets at 8 4.69 (s, IH) and 4.76 (s, IH) ppm (Table 12) plus an additional 14 mass units ("CH2) in the mass spectrum indicated the presence of an olefinic-methylene functionality in the side chain as seen in compounds 117 and 119. Subtracting the two carbons assigned to the methyl ether and olefinic methylene from the total of 28 carbons in the molecular formula of compound F, leaves a total of only 26 carbon atoms. Therefore compound F cannot have a standard steroidal skeleton. Further examina-tion of the mass spectrum of compound F 121 revealed a strong peak at m/z 344.2351 (calcd. 344.2353) indicating a loss of 84 daltons (C 6H 1 2) 112 -114 assigned to loss of a truncated side chain via a McLafferty rearrange-ment (Scheme 28). A similar fragmentation pattern was observed for compound E. Scheme 28: MS fragmentation of compound F 121 m/z A28 (5 7.) m/z 3U (12 •/.) This evidence allowed the assignment of shortened side chains in both compounds E and F. Based on the spectroscopic data accumulated in these two compounds, tentative structures 120 and 121 were assigned pending further isolation of sufficient material which would f a c i l i t a t e an unambiguous assignment of their structures. 115 3D. Biosynthesis of Sponge Metabolites A biosynthetic proposal for the formation of the new s t e r o i d s isolated from Anthoarcuata graceae could logically begin w i t h A 2 4-cholesterol. A proposed biogenetic pathway for the two A 4-3,6-diketosteroids 116 and 117 is outlined in Scheme 29. The side chain found in compounds 117 and 119 could be formed by biomethylation with S-adenosylmethionine (SAM) and a l l y l i c rearrangement (steps 2 and 3). A l l y l i c rearrangement of the double bond (step 4) followed by oxidation of the 3^-hydroxyl group (step 5) and a l l y l i c hydroxylation and oxidation at the C-6 position (steps 6, 7) represents a possible biogenetic route to compounds 116 and 117. A mixture of steroids including 4,7,22-triene-3,6-diketones 124 have been isolated by Malorni et a l . ^ 4 from the marine sponge, Raphidos- t i l a incisa. collected off Zlarin, Yugoslavia, and cholesta-4,7-diene-3,6-dione (125), was isolated by Kinnear et al.^-> from the i n s e c t , R 0 124 R=H 125 R=H 2 116 -Scheme 29: Biosynthetic proposal f o r compounds A and B - 117 -C a l l i p h o r a s t y g i a . I t appears, however, that compounds 116 and 117 are the only examples of n a t u r a l l y occurring A 4-3,6-diketosteroids from marine sources. A possible biosynthetic pathway to the anthosterones A (118) and B (119) proceeds through a diosphenol intermediate as seen i n Scheme 30. I f the diosphenol i s converted to the a-diketone tautomer (Step 1), contraction of the A r i n g can occur v i a a benzylic a c i d rearrangement 8^ (Step 2) to give the a-hydroxy carboxylic a c i d intermediate, which on hydroxylation and oxidation (Step 3), followed by methylation (Step 4) would form the common nucleus found i n natural products 118 and 119. I t appears from a l i t e r a t u r e search that the nucleus found i n compounds 118 and 119 i s unprecedented. I t i s not completely c l e a r yet whether these unusual A-nor steroids are metabolites elaborated by the sponge or a r t i f a c t s formed during i s o l a t i o n . An ethanol extract was examined i n an attempt to i s o l a t e the e t h y l ester d e r i v a t i v e . This experiment was inconclusive due to i n s u f f i c i e n t material. A proposed pathway to the diosphenols 120 and 121 i s o u t l i n e d i n Scheme 31 with the a d d i t i o n of methanol to the C-6 p o s i t i o n . Examples of side chains shortened by one carbon are known from marine organisms. 8 7 A previous b i o s y n t h e t i c proposal involved the methylation of C-24 with SAM, followed by oxidative degradation to lose C26/27 of the side chain (Scheme 3 2 ) . 8 8 Subsequent methylations and reductions could y i e l d the side chains found i n the diosphenols E and F (Scheme 32). The side chains proposed f o r compounds 120 and 121 appear to be the f i r s t n a t u r a l l y occurring examples of these side chains. 118 -Scheme 30: B i o s y n t h e t i c proposal f o r A-nor s t e r o i d s 118, 119 - 119 -120 G. EXPERIMENTAL The -"-H NMR spectra were recorded on e i t h e r the Bruker-WH-400, Bruker WP-80, N i c o l e t - O x f o r d 270 or Var i a n XL-300 spectrometers. Tetramethylsilane (6 = 0) was employed as the i n t e r n a l standard f o r ^ H NMR spec t r a and CDC1 3 (5 = 77.0 ppm) or DMS0-d6 (6 - 39.5 ppm) were used both as i n t e r n a l standards as w e l l as solve n t s f o r 1 3 C NMR spectra unless otherwise i n d i c a t e d . Low r e s o l u t i o n and high r e s o l u t i o n e l e c t r o n impact mass spectra were recorded on Kratos MS-59 and MS-50 spectrometers r e s p e c t i v e l y . I n f r a r e d s p e c t r a were recorded on a Perkin-Elmer 1710 FT spectrometer and UV ab s o r p t i o n spectra were measured on a Bausch and Lomb Spectronic 2000 spectrometer. O p t i c a l r o t a t i o n s were measured on Perkin-Elmer model 141 po l a r i m e t e r u s i n g a 10 cm c e l l , while uncorrected m e l t i n g p o i n t s were determined on a Fisher-Johns m e l t i n g p o i n t apparatus. HPLC was c a r r i e d out on e i t h e r a Perkin-Elmer Series 2 instrument equipped w i t h a Perkin-Elmer LC-55 UV detector or a Waters model 501 system equipped w i t h a Waters 440 dual wavelength detector f o r peak d e t e c t i o n . The HPLC column used was the Whatman Magnum-9 0DS-3 reverse phase p r e p a r a t i v e column. The solvents used f o r HPLC were BDH Ombisolve or F i s h e r HPLC grade and the water used was g l a s s - d i s t i l l e d . A l l other sol v e n t s used were at l e a s t reagent grade unless otherwise i n d i c a t e d . S i l i c a g e l types used were Merck s i l i c a g e l 60 PF-254 f o r prepara-t i v e TLC, Merck s i l i c a g e l 60 230-400 mesh f o r f l a s h chromatography and Merck s i l i c a g e l 60 PF-254 w i t h CaSO^ 1/2H20 f o r r a d i a l TLC. A l l R f 121 v a l u e s were c a l c u l a t e d on a n a l y t i c a l TLC p l a t e s u s i n g M a c h e r e y - N a g e l S i l G/UV 254 p r e c o a t e d s h e e t s .25 mm t h i c k . P r e p a r a t i o n o f m e t h y l a t e d 4 - m e t h y l - 2 - n i t r o p h e n o l 41 To a s t i r r e d s u s p e n s i o n o f p o t a s s i u m c a r b o n a t e ( 1 . 5 0 0 g , 1 0 . 9 mmol) i n d i s t i l l e d a c e t o n e (7 ml ) i n a 25 ml r o u n d b o t t o m f l a s k was added 4 - m e t h y l - 2 - n i t r o p h e n o l ( 1 . 0 0 0 g , 6 .5 mmol) d i s s o l v e d i n a c e t o n e (5 m l ) . The r e s u l t i n g y e l l o w s o l u t i o n t u r n e d c o m p l e t e l y r e d i n a b o u t 2 m i n u t e s . A f t e r t h i s , m e t h y l i o d i d e ( 1 . 8 0 0 g , 1 2 . 7 mmol) was added d r o p w i s e and the r e a c t i o n m i x t u r e was r e f l u x e d f o r a p p r o x i m a t e l y 40 m i n u t e s w h i l e b e i n g m o n i t o r e d b y TLC ( 1 0 : 9 0 e t h y l a c e t a t e / p e t r o l e u m e t h e r ) . The r e d r e a c t i o n m i x t u r e was vacuum f i l t e r e d and the r e s u l t i n g f i l t r a t e was p o u r e d i n t o a s e p a r a t o r y f u n n e l and p a r t i t i o n e d be tween w a t e r (25 ml ) and c h l o r o f o r m (4 x 30 m l ) . The c h l o r o f o r m l a y e r s were c o m b i n e d , d r i e d o v e r a n h y d r o u s Na2S04 , and f i l t e r e d . E v a p o r a t i o n i n v a c u o y i e l d e d a y e l l o w gum w h i c h on p u r i f i c a t i o n by p r e p a r a t i v e TLC ( 1 0 : 9 0 e t h y l a c e t a t e / p e t r o l e u m e t h e r ) gave compound 4 1 , 4 5 0 . 6 mg (41%) as a y e l l o w o i l : X H NMR (80 MHz, C D C 1 3 ) 5 2 . 3 5 ( s , 3 H ) , 3 . 95 ( s , 3 H ) , 6 .99 ( d , J -9 H z , I H ) , 7 . 3 5 ( d d , J - 9, 2 H z , I H ) , 7 . 6 0 ( d , J - 2 H z , IH) ppm; HRMS, m/z o b s e r v e d 1 6 7 . 0 5 8 3 , r e q u i r e d f o r C 8 H Q N 0 3 1 6 7 . 0 5 8 3 ; MS m/z ( r e l a t i v e i n t e n s i t y ) 167 ( 7 8 ) , 137 ( 4 9 ) , 120 ( 8 2 ) , 105 ( 2 7 ) , 91 ( 1 0 0 ) , 77 ( 5 0 ) , 65 ( 7 5 ) . 122 Preparation of Brominated Compound 51 Purified N-brominosuccinimide (0.155 g, .87 mmol) was added to a refluxing solution of 41 (.124 g, .81 mmol) dissolved in carbon tetrachloride (5 ml) in a 10 ml round bottom flask. The refluxing mixture was irradiated for 1 hr with a 150 W tungsten light bulb. After cooling, the mixture was fi l t e r e d and the f i l t r a t e was concentrated in vacuo. The resulting yellow heavy o i l was purified by preparative TLC (3:10 ethyl acetate/hexanes) giving a clear yellow o i l (Rf. 51), 0.089 g (45%): MS, m/z (relative intensity) 247 (2), 245 (3), 166 (65), 134 (51), 120 (25), 91 (34), 77 (62). Preparation of methylated phidolopin 53 Purified compound 51 (0.080 g, 0.36 mmol) was dissolved in tetrahydrofuran (3 ml) and added dropwise to a sti r r i n g solution of theophylline (0.051 g, 0.28 mmol) in 0.1 M NaOH (2.5 ml). After addi-tion was complete, the reaction mixture was refluxed for 1 hr. At the end of this time the reaction mixture was cooled and partitioned between d i s t i l l e d water (15 ml) and chl oroform (4 x 20 ml). The chloroform layer was dried over anhydrous Na2S04 and fil t e r e d . Evaporation of the solvent followed by purification by preparative TLC (ethyl acetate) yielded 0.043 g (81%) of compound 53 as a white solid: 1H NMR (80 MHz, CDC13) 3.43 (s, 3H), 3.58 (s, 3H), 5.48 (s, 2H), 7.00 (d, J - 9 Hz, IH), 7.38 (dd, J = 9, 2 Hz, IH), 7.68 (d, J - 2 Hz, IH) ppm; HRMS, m/z 123 -observed 345.1075, required for C 1 5H 15N 50 5 345.1075; MS, m/z ( r e l a t i v e i n t e n s i t y ) 345 (M, 7), 180 (23), 166 (100), 91 (21), 77 (14). Preparation of the MOM protected 4-methyl-2-nitrophenol 42 To a s t i r r e d suspension of anhydrous potassium carbonate (0.897 g, 6.5 mmol) i n d i s t i l l e d acetone (20 ml) under a nitrogen atmosphere i n a 50 mL round bottom f l a s k was added 4-methyl-2-nitrophenol (1.000 g, 6.5 mmol) dissolv e d i n acetone (5 ml). The r e s u l t i n g yellow s o l u t i o n turned deep red a f t e r 15-20 minutes at room temperature. Anhydrous chloro-methylmethyl ether (1.50 ml, 19.5 mmol) was added dropwise and the resu l t a n t yellow s o l u t i o n was s t i r r e d f o r 10 minutes. The reaction mixture was f i l t e r e d and the f i l t r a t e was added to a 150 ml separative funnel containing 10 ml d i s t i l l e d water. The aqueous layer was repeatedly extracted with portions of chloroform (25 ml) u n t i l the yellow color had disappeared. The combined organic layer was dried over anhydrous Na2S0^ and f i l t e r e d . Evaporation of the solvent i n vacuo followed by preparative TLC (1:10 ethylacetate/hexanes) y i e l d e d 0.930 g (73%) of compound 42 as a yellow o i l : *H NMR (80 MHz, CDCl 3) 5 2.35-3.53 (s, 3H), 5.25 (s, 2H), 7.13 (bd, J - 9 Hz, IH), 7.30 (dd, J = 9, 2 Hz, IH), 7.56 (d, J - 2 Hz, IH) ppm; HRMS, m/s observed 197.0693, required f o r C 9H 1 1N0 4 197.0688; MS m/z ( r e l a t i v e i n t e n s i t y ) 197 ( 5 5 ) , 167 (34), 166 (2.5), 136 (11), 45 (100). 1 2 4 Preparation of MOM Phidolopin 55 Purified N-brominosuccinimide (.242 g, 1.36 mmol) was added to a refluxing solution of 42 (.0891 g, .45 mmol) dissolved in carbon tetrachloride (5 ml) in a 10 ml round bottom flask. The refluxing mixture was irradiated for 15 minutes with a 250 W sun lamp. After cooling, the mixture was f i l t e r e d and the f i l t r a t e was concentrated in vacuo. The resulting brown solid was dissolved in tetrahydrofuran (5 ml) and added dropwise to a solution of theophylline (.0823 g, .045 mmol) and .1 M sodium hydroxide (2.5 ml). After addition was complete, the reaction mixture was stirred at room temperature for 36 hours. At the end of this time the reaction mixture was partitioned between d i s t i l l e d water (15 ml) and chloroform (4 x 20 ml). The chloroform layer was dried over anhydrous Na2S04 and filt e r e d . Evaporation of the solvent followed by purification by preparative TLC (ethyl acetate) gave .534 g (32%) of pure 55 as a white solid: lH NMR (80 MHz, CDC13) 6 3.40 (s, 3H), 3.50 (s, 3H), 3.71 (s, 3H), 5.25 (s, 2H), 5.46 (s, 2H), 7.28 (bd, J = 8.5 Hz, IH), 7.53 (dd, J - 8.5, 2 Hz, IH), 7.63 (s, IH), 7.75 (d, J = 2.1 Hz, IH) ppm; HRMS, m/z observed 375.1178, required for C16 H17 N5°6 375.1180; MS m/z (relative intensity) 375 (8), 393 (M+ + H20, 2), 330 (6), 313 (21), 258 (27), 209 (32), 180 (100), 95 (79). 125 Preparation of Phidolopin (36) Deprotection of the MOM derivative was achieved by refluxing 54 (.025 g, .067 mmol) dissolved in chloroform (3 ml) in 50% acetic acid plus one drop of concentrated sulphuric acid for 1 hour. Progress of the reaction was monitored via TLC (ethyl acetate). The yellow reaction mixture was partitioned between water (10 ml) and chloroform (4 x 20 ml). The chloroform layer was dried over sodium sulphate and filte r e d . Evaporation of the solvent in vacuo, followed by purification by prepar-ative TLC (ethyl acetate) gave .0215 g (97%) of Phidolopin (R f .19), yellow needles: mp 212-213°C (CHCI3); XH NMR (80 MHz, CDCl3-DMS0-d6) 6 3.41 (s, 3H), 3.59 (s, 3H), 5.48 (s, 2H), 7.16 (bd, H = 8.5 Hz, IH), 7.63 (dd, J - 1.9, 8.5 Hz, IH), 7.65 (s, IH), 8.06 (d, J - 1.9 Hz, IH), 10.6 (s, IH) ppm; HRMS, m/z observed 331.0908, required for C 1 4H 1 3N 50 5 331.0917; EI-LRMS m/z (relative intensity) 331 (M+, 91), 313 (20), 180 (100), 152 (29), 123 (21), 95 (30); UV (CHCI3) 355 (e 1900), 275.8 (e 9100): 1 3C NMR (750 MHz, CDCl3-DMS0-d6) 5 27.62, 29.46, 47.99, 105.89, 119.71, 124.94, 127.88, 135.48, 142.21, 148.58, 151.11, 152.56, 154.57 ppm. Preparation of MOM desmethylphidolopin 56 Purified N-brominosuccinimide (1.470 g, 8.25 mmol) was added to a refluxing solution of 42 (.41 g, 2.1 mmol) dissolved in carbon tetrachloride (5 ml) in a 10 ml round bottom flask. The refluxing - 126 mixture was irradiated for 90 minutes with a 250 W sun lamp. After cooling, the mixture was f i l t e r e d and the f i l t r a t e was concentrated in vacuo. The resulting brown solid was dissolved in tetrahydrofuran (5 ml) and added dropwise to a solution of 3-methylxanthine (.3 g, 1.8 mmol) and 0.1 M NaOH (3.5 ml) which was stirred at room temperature for 16 hours. At the end of this time, the reaction mixture was partitioned between d i s t i l l e d water (15 ml) and chloroform (4 x 25 ml). The chloro-form layer was dried over anhydrous Na2S04 and f i l t e r e d . Evaporation of the solvent followed by purification by radial preparative TLC (ethyl acetate) gave 0.026 g (25%) of pure 56 as a white solid: 1H NMR (80 MHz, CDC13) 6 3^35 (s, 3H), 3.44 (s, 3H), 5.44 (s, 2H), 7.38 (d, J - 9 Hz, IH), 7.66 (dd, J = 9, 2 Hz, IH), 7.95 (d, J - 5 Hz, IH), 8.26 (s, IH) ppm; HRMS, m/z observed 361.1025, required for C^H^^Og 361.1495; EI-LRMS m/z (relative intensity) 361 (76), 343 (31), 329 (46), 299 (38), 166 (75), 152 (34), 134 (61), 121 (30), 105 (38), 94 (63), 77 (95), 51 (100). Preparation of Desmethylphidolopin (37) Deprotection of the MOM derivative was achieved by refluxing 55 (0.026 g, 0.071 mmol) dissolved in chloroform (3 ml) in 50% acetic acid plus one drop of concentrated sulphuric acid for 1 hr. Progress of the reaction was monitored via TLC (ethyl acetate).' The yellow reaction mixture was partitioned between water (10 ml) and chloroform (4 x 20 ml). The chloroform layer was dried over anhydrous Na2S0^ and filtered. - 127 -Evaporation of the solvent in vacuo, followed by purification by pre-parative TLC (ethyl acetate) gave .024 g (94%) of desmethylphidolopin (37) (R f .15) as a yellow solid: XH NMR (80 MHz, CDC13) 8 3.46 (s, 3H), 5.45 (s, 2H), 7.10 (d, J - 8.5 Hz, IH), 7.68 (dd, J - 8.5, 1.9 Hz, IH), 7.82 (s, IH), 8.05 (d, J - 1.9 Hz, IH), 11.03 (s, IH); HRMS, m/z observed 317.0760, required for C 1 3H 1 1N 50 5 317.0761; EI-LRMS m/z (relative intensity) 317 (M+, 2), 299 (10), 166 (100), 152 (49), 123 (45), 106 (37), 95 (55), 77 (53), 68 (80); 1 3C NMR (75 MHz, CDCl3-DMS0-d6) 8 28.5, 47.9, 106.3, 119.9, 125.2, 127.6, 135.2, 142.3, 150.4, 151.1, 152.8, 154.9 ppm. 128 BRYOZOANS C o l l e c t i o n Data B r y o z o a n s were c o l l e c t e d u s i n g SCUBA a t v a r i o u s l o c a t i o n s i n B a r k l e y S o u n d , B . C . a t d e p t h s f rom 2 to 10 m. I m m e d i a t e l y a f t e r c o l l e c -t i o n , the whole a n i m a l s were immersed i n m e t h a n o l and s t o r e d f o r one to t h r e e days a t room t e m p e r a t u r e . I f the a n i m a l s were n o t i m m e d i a t e l y worked u p , t h e y were s t o r e d a t l o w e r t e m p e r a t u r e ( a b o u t 2 C C ) u n t i l u s e d , n o r m a l l y w i t h i n one month . E x t r a c t i o n and C h r o m a t o g r a p h i c S e p a r a t i o n 1. D i a p e r o e c i a c a l i f o r n i c a ( d ' O r n i g n y 1892) D i a p e r o e c i a c a l i f o r n i c a (653 g d r i e d w e i g h t a f t e r e x t r a c t i o n ) was g r o u n d i n a W a r i n g b l e n d e r w i t h the m e t h a n o l (1 L) u s e d f o r e x t r a c t i o n o f the whole a n i m a l s . The s u s p e n s i o n o f g r o u n d b r y o z o a n s was f i l t e r e d t h r o u g h c e l i t e , i n v a c u o , y i e l d i n g a r e d d i s h brown aqueous m e t h a n o l i c f i l t r a t e w h i c h was c o n c e n t r a t e d t o a p p r o x i m a t e l y 300 ml and p a r t i t i o n e d be tween b r i n e (200 ml) and e t h y l a c e t a t e (3 x 400 m l ) . The combined d a r k r e d e t h y l a c e t a t e l a y e r s were d r i e d o v e r a n h y d r o u s Na2S0^. F i l t r a t i o n , f o l l o w e d by e v a p o r a t i o n , i n v a c u o , gave 5 .8 g (.89%) o f a d a r k r e d c r u d e o i l . F l a s h c h r o m a t o g r a p h i c f r a c t i o n a t i o n (40 mm d i a m e t e r - 129 -column, 15 cm s i l i c a gel, step gradient 5% ethyl acetate/hexanes to 100% ethyl acetate) yielded fractions containing fats, sterols, pigments as well as three strongly absorbing UV bands on TLC (100% ethyl acetate). Purification of the most non polar band by column chromatography, Sephadex LH-20 (7:3 methanol/chloroform) yielded a strongly retained yellow band corresponding to 4-hydroxymethyl-2-nitrophenol (38) (4.5 mg, .007%, Rf. 35). Column chromatography of the next most polar fraction using Sephadex LH-20 (7:3 methanol/chloroform) afforded a strongly retained yellow band corresponding to phidolopin (36) (1.0 mg, <.001%, Rf. 24). Purification of the most polar band in a similar fashion gave desmethylphidolopin (37) (3.7 mg, <.001%, Rf. 15). Phidolopin (36): mp 211-212°C (CHC13); UV ( C H 3 C N ) A m a x 351 nm (e 3300), 275 (e 16800); % NMR (270 MHz, C D C I 3 ) 8 10.55 (s, IH, exchanges with D 20), 8.09 (d, J - 2.5 Hz, IH), 7.63 (s, IH), 7.61 (dd, J = 2.5, 8.5 Hz, IH), 7.17 (d, J - 8.5 Hz, IH), 5.46 (s, 2H), 3.59 (s, 3H), 3.40 (s, 3H); HRMS observed m/z 331.0914, required for C 1 4H 13N 50 5 331.0917; LRMS, m/z (relative intensity) 331 (14), 313 (25), 180 (100), 152 (38). Desmethylphidolopin (37): ^-H NMR (80 MHz, C D C I 3 ) 5 10.60 (s, IH), 8.06 (d, J = 2 Hz), 7.63 (s, IH), 7.59 (dd, J = 2, 9 Hz), 7.13 (d, J - 9 Hz), 5.39 (s, 2H), 3.51 (s, 3H) ppm; HRMS observed m/z 317.0760, calculated for C 1 3H 1 1N 50 5 317.0761; LRMS, m/z (relative intensity) 317 (4), 299 - 130 (12), 166 (100), 152 (52), 123 (45), 106 (37), 95 (55), 77 (54), 68 (80), 51 (71). 4-hydroxymethyl-2-nitrophenol (38): 1H NMR (80 MHz, CDCI3) 6 10.53 (s, IH), 8.09 (d, J - 2 Hz), 7.58 (dd, J - 2, 9 Hz, IH), 4.69 (s, 2H) ppm;, HRMS m/z 169.0375, calculated for C 7H 7N0 4 169.0375; LRMS, m/z (relative intensity) 169 (100), 152 (11), 123 (25), 122 (11), 106 (16), 95 (15), 77 (25), 65 (45). 4-methoxymethyl-2-nitrophenol (39): XH NMR (80 MHz, CDCI3) 5 10.58 (s, IH), 8.10 (d, J - 2 Hz, IH), 7.60 (dd, J - 2, 9 Hz, IH), 4.43 (s, 2H), 3.44 (s, 3H) ppm; HRMS observed m/z 183.0535, calculated for CgHqN04 183.0532; LRMS, m/z (relative intensity) 183 (59), 182 (29), 152 (100), 141 (25), 136 (21), 123 (12), 106 (39), 105 (14), 77 (24), 65 (12). 2. The remaining bryozoans, Heteropora alaskensis. Hippodiplosia  insculpta. T r i c e l l a r i a ternata. were extracted as described above for IL_ californica. The results are shown in the Table 13. - 131 Table 13: Nitrophenols from Northeast Pacific Bryozoans Organism *Crude EtOAc Yield mg (%) Extract 36 37 38 39 1 Diaperoecia californica 5.8 ( -89) 1.0 ( .001) 3.7 4.5 (<.001) ( .007) -2 Heteropora alaskensis 5.7 (1.20) - 1.9 (<.001) -3 Hippodiplosia insculpta 3.7 (1.50) - 3.6 (<.001) 2.4 (<.001) 4 T r i c e l l a r i a ternata 5.2 ( -72) - - 3.4 (<.001) g (% dry weight after extraction) - 132 -ANTHOARCUATA GRACEAE (Bakus 1966) Collection Data Anthoarcuata graceae was collected during a l l seasons at various locations in Barkley Sound, B.C. at depths of 1 to 5 metres. Immedi-ately after collection, the sponge was immersed in methanol or ethanol and stored at room temperature for up to three days. If the sponge was not worked up immediately, i t was stored at low temperature (4-(-5)"C) u n t i l used (typically within 2 weeks). Extraction and Chromatographic Separation During the course of study on the extracts of the marine sponge Anthoarcuata graceae. a number of collections were made yielding l i t t l e or no observed variation in metabolites. Therefore, the following represents a typical procedure. After storage at room temperature for 2 days, the aqueous metha-nolic layer was decanted and stored at room temperature while the sponge, approximately 1200 g (dry weight after extraction) was further soaked in dichloromethane (6 L) for 12 hours before being transferred in dichloromethane into a Waring blender and ground down into a slurry. This crude slurry was vacuum filte r e d , along with the i n i t i a l aqueous methanolic layer, through celite and the deep red f i l t r a t e was concen-133 trated to about 250 ml before being partitioned between brine (15 ml) and dichloromethane (4 x 30 ml). The combined dichloromethane extracts were then dried over anhydrous Na2S04. The sponge solids from the f i l t r a t i o n were transferred back into the original collection jars and soaked in dichloromethane three more times at 1 day intervals. The same procedure of f i l t r a t i o n , partitioning of f i l t r a t e and drying the organic layers over anhydrous Na2S04 was repeated each day. The combined dichloromethane extracts were f i l t e r e d and concen-trated in vacuo to yield a crude dark red o i l , 46.9 g (3.9% of dry sponge after extraction). The o i l was fractionated by flash chromatog-raphy (50 mm diameter column, 16 cm s i l i c a gel, step gradient of 100% hexanes to 30% ethyl acetate/hexanes) to yield crude fractions contain-ing fats, pigments, carotenoids and a mixture of steroids detected by analytical TLC (50% ethyl acetate/hexanes). The combined steroid containing fractions (charring a bright pink colour with 50% H2SO4 spray reagent) were pooled and evaporated to yield 8.2 g (.68%) of a s t i l l crude red o i l . Further purification of this complex mixture of fats, carotenoids and pigments in addition to the steroidal components was carried out using repeated column chromatography (20 mm diameter column, packed with 1 m of Sephadex LH-20, 55% methanol/chloroform as running solvent) to yield fractions containing fats, carotenoids and steroids. The s t i l l impure steroidal fraction from column chromatography was further purified by preparative reverse phase HPLC (10% water/aceto-n i t r i l e ) to yield two major peaks (retention times 40.6 min. and 47.4 min., flow rate of 2.8 ml/min., UV A m a x 254 nm) which each were found to possess 3 novel minor steroids as detected by analytical TLC analysis. 134 F i n a l separation and p u r i f i c a t i o n of the three components from each HPLC peak were c a r r i e d out by preparative TLC (5% methanol/ dichloromethane) y i e l d i n g compound A ( .0064 g, .0005%, R f .74), compound D ( .0073 g , .0006%, R f . 6 4 ) , compound F ( .0037 g, .0003%, R f .42) from the second peak with retention time 47.4 min. Compound 116: mp 92-93°C (CH3CN); UV 248 .9 nm (e 11600) ; IR (CHCI3 cast) 3031, 2956, 2871, 1686, 1643, 1606 cm - 1; -^H NMR (270 MHz, CDC13) 6 0.73 (s, 3H), 0.95 (d, J - 6 .0 H z , 3H), 1.17 (s, 3H), 1.61 (s, 3H), 1.69 (s, 3H), 5 .09 ( t , J - 6 .4 H z , I H ) , 6 .19 (s, IH) ppm; 1 3 C NMR (75 MHz, CDCI3) 6 202.36, 1 9 9 . 5 6 , 1 6 1 . 0 6 , 1 2 7 . 0 9 , 1 2 5 . 4 8 , 1 2 4 . 9 9 , 5 6 . 5 5 , 5 5 . 9 1 , 5 0 . 9 9 , 4 6 . 8 3 , 42.57, 39 .14, 35 .98, 35 .56, 35 .50, 34 .22, 34 .17, 3 3 . 9 8 , 2 7 . 9 9 , 25.74, 2 4 . 6 9 , 2 3 . 9 8 , 20 .89, 1 8 . 5 5 , 1 7 . 5 1 , 11 .91 ppm; E I -HRMS, m/z observed 3 9 6 . 3 0 3 4 , required f o r C27H40O2 396 .3030 ; EI -LRMS m/z ( r e l a t i v e i n t e n s i t y ) 396 ( 2 6 ) , 381 ( 1 9 ) , 312 (68), 283 ( 4 6 ) , 270 ( 1 3 ) , 257 ( 1 9 ) , 243 ( 1 8 ) , 137 ( 5 2 ) , 105 (20), 95 ( 5 5 ) , 81 ( 5 2 ) , 69 ( 9 2 ) , 55 (100). Compound 117: mp 111-112°C (CH3CN): UV (CH3CN) 248 .6 nm (e 12500) ; X H NMR (270 MHz, CDCI3) 6 0.73 (s, 3H), 0 .85 (d, J - 7 .0 H z , 3H), 1.02 (d, J - 8 .0 H z , 3H), 1.17 (s, 3H), 3.49 (s, 3H), 4 . 6 5 (s, IH ) , 4.73 (s, I H ) , 6 .17 (s, IH) ppm; 1 3 C NMR (75 MHz, CDCI3) 202.36, 1 9 9 . 5 6 , 1 6 1 . 0 8 , 1 5 6 . 6 5 , 125.44, 1 0 6 . 0 6 , 5 6 . 6 1 , 55.77, 5 0 . 9 3 , 46.79, 4 2 . 5 5 , 39.10, 35 .62, 35 .51, 34 .50, 34 .18, 33 .96, 3 0 . 8 9 , 2 9 . 7 0 , 2 7 . 9 8 , 2 3 . 9 5 , 2 2 . 7 0 , 21.99, 21 .84, 20.86, 1 8 . 6 2 , 1 7 . 5 1 , 11.88 ppm; E I -HRMS, m/z observed 4 1 0 . 3 1 8 2 , required for C 28H4 20 2 4 1 0 . 3 1 8 7 ; E I - L R M S , m/e - 135 -(relative intensity) 410 (36), 327 (76), 297 (19), 283 (49), 257 (53), 137 (800, 109 (42), 95 (52), 81 (62), 69 (83), 55 (100). Compound 118: mp 142-143°C (CH3CN); UV (CH3CN) A m a x 250.2 nm (e 12500); IR (CHCI3 cast) 3552, 3021, 2951, 2869, 1744, 1717, 1 6 48 cm-1; 1H NMR (400 MHz, CDCI3) S 0.73 (s, 3H), 0.96 (d, J = 6.7 Hz, 3H), 1.21 (s, 3H), 1.62 (s, 3H), 1.62 (s, 3H), 1.69 (s, 3H), 1.88 (m, IH), 2.08 (d, J = 13.3 Hz, IH), 2.19 (d, J = 13.3 Hz, IH), 2.41 (m, IH), 3.79 (s, 3H), 5.10 (t, J = 8.0 Hz, IH), 6.73 (t, J - 3.3 Hz, IH) ppm; 1 3C NMR (75 MHz, CDCI3) S 200.91, 173.37, 145.10, 136.28, 131.05, 125.09, 79.90, 56.26, 56.02, 55.91, 53.39, 49.66, 42.94, 39.92, 39.35, 36.06, 35.55, 32.32, 32.21, 28.11, 25.75, 24.74, 24.35, 22.11, 21.79, 18.66, 17.66, 12.03 ppm; EI-HRMS, m/z observed 442.3083, required for C 2 8H 4204 442.3079; EI-LRMS m/z (relative intensity) 442 (79), 427 (24), 383 (10) , 358 (23), 340 (18), 329 (41), 311 (19), 298 (30), 283 (7), 269 (11) , 135 (32), 147 (22), 107 (41), 95 (58), 81 (49), 69 (100), 55 (68). Compound 119: Needles; mp (CH3CN) 155-157°C; UV (CH3CN) A m a x 249.3 (e 12034); IR (CHCI3, cast), 3022, 2929, 2856, 1744, 1717, 1643 cm"1; XH NMR (400 MHz, CDCI3) S 0.73 (s, 3H), 0.97 (d, J = 6.5 Hz, 3H), 1.04 (d, J = 6.5 Hz, 3H), 1.20 (s, 3H), 1.89 (m, IH), 2.10 (d, J - 13.5 Hz, IH), 2.18 (d, J - 13.5 Hz, IH), 2.39 (m, IH), 3.77 (s, 3H), 4.65 (s, IH), 4.71 (s, IH), 6.73 (t, J = 2.5 Hz, IH) ppm; 1 3C NMR (75 MHz, CDCI3) 6 200.91, 173.36, 156.79, 145.08, 136.29, 106.01, 79.89, 56.25, 55.93, 53.40, 49.64, 46.68, 42.95, 39.93, 39.34, 35.72, 34.64, 33.81, 32.32, 32.21, 30.99, 28.13, 24.34, 22.11, 22.01, 21.87, 21.79, 18.74, 12.03 - 136 -ppm; EI-HRMS, m/z observed 456.3236, required for C29H44O4 456.3241; EI-LRMS m/z (relative intensity) 456 (2), 396 (2), 367 (23), 353 (5), 298 (48), 284 (10), 269 (65), 147 (35), 124 (56), 109 (49), 95 (73), 81 (68), 69 (89), 55 (100). Compound 120: 1H NMR (400 MHz, CDCI3) 5 0.73 (s, 3H), 0.93 (d, J = 6 Hz, 3H), 1.28 (d, J = 8 Hz, 3H), 1.52 (s, 3H), 1.69 (s, 3H), 2.18 (d, J - 16 Hz, IH), 2.25 (d, J = 16 Hz, IH), 3.29 (s, 3H), 4.52 (t, J - 3 Hz, IH), 5.09 (t, J - 7 Hz, IH), 5.28 (s, IH) ppm; EI-HRMS, m/z observed 414.3149, required for C27H42O3 414.3136; EI-LRMS m/z (relative intensity) 414 (19), 382 (28), 268 (10), 330 (16), 298 (15), 269 (29), 245 (30), 161 (26), 138 (34), 109 (34), 97 (52), 81 (49); 69 (100), 55 (81); UV 264.7 nm (e 10971, c - .04) + NaOH (304.7 nm, e 9731). Compound 121: XH NMR (400 MHz, CDCI3) S 0.73 (s, 3H), 0.94 (d, J = 8 Hz, 3H), 1.03 (d, J - 7 Hz, 6H), 1.31 (s, 3H), 2.17 (d, J = 16 Hz, IH), 2.29 (d, J - 16 Hz, IH), 3.33 (s, 3H), 4.57 (t, J - 2 Hz, IH), 4.69 (s, IH), 4.76 (s, IH), 5.28 (s, IH) ppm; EI-HRMS, m/z observed 428.3284 required for C28H40O3 428.3291; EI-LRMS m/z (relative intensity) 428 (5), 396 (13), 344 (12), 312 (14), 300 (12), 271 (24), 161 (30), 138 (37), 107 (38), 95 (52), 81 (61), 69 (95), 55 (100). - 137 -Preparation of 4-ene-3,6-dione 122 by Jones oxidation The Jones reagent was prepared by dissolving chromium trioxide (26.72 g, 270 mmol) in 50 ml d i s t i l l e d water in a 100 ml beaker. The beaker was immersed in an ice water bath and concentrated sulfuric acid 911.5 ml, 200 mmol) was added followed by enough d i s t i l l e d water to bring the total volume fo 100 ml. A solution of cholesterol (1.00 g, 2.6 mmol dissolved in 30 ml acetone was stirred in a 50 ml round bottom flask at 0°C in an ice water bath before 2 ml of the previously prepared cold oxidation reagent was added at a rate to maintain a reaction mixture temperature of around 20°C. The st i r r i n g was maintained for 3 hours, with the reaction mixture turning yellowish from the original deep red colour. After 3 hours, 10 ml of methanol was added to destroy excess reagent, and the reaction mixture was f i l t e r e d through celite. The resulting light brown solution was placed into a 150 ml separatory funnel and extracted with chloroform (4 x 50 ml). The organic layer was further washed with 50 ml of brine and dried over anhydrous magnesium sulfate. F i l t r a t i o n , followed by evaporation in vacuo yields a light brown gum (462 mg) which on preparative TLC yuielded one highly absorb-ing UV band at Rf .50 (30% ethyl acetate/hexanes) which based on spec-t r a l data corresponded to the ene-dione 122, 337.9 mg (32.7%). Compound 122: mp 132-134°C (CH3CN); IR (CHC13 cast) 2953, 2871, 1685, 1606 cm'1; XH NMR (270 MHz, CDCI3) S 0.74 (s, 3H), 0.87 (d, J = 6 Hz, 6H), 0.94 (d, J = 6 Hz, 3H), 1.19 (s, 3H), 6.19 (s, IH) ppm; 1 3C NMR (75 MHz, CDCI3) 5 202.25, 199.40, 160.98, 125.33, 56.42, 55.82, - 138 -50.83, 46.71, 39.70, 39.36, 39.01, 35.57, 35.40, 34.09, 33.87, 27.91, 23.86, 23.69, 22.75, 20.77, 18.55, 17.40, 11.79 ppm; EI-HRMS, m/z observed 398.3186 required for C21Y[M°2 398.3187; EI-LRMS m/z (relative intensity) 392 (2), 383 (14), 370 (6), 343 (2), 329 (6), 243 (40), 147 (24), 135 (33), 124 (35), 105 (37), 91 (65), 79 (64), 67 (55), 55 (100). Anthosterone B-acetate (123) 2.8 mg (.006 mmol) of Compound D 119 was stirred in pyridine and acetic anhydride (2:1) in a 10 ml round bottom flask for 12 hours at room temperature. Evaporation of the solvent and excess reagent gave acetylated product 123, as a white solid, in quantitative yield. Compound 123: lW NMR (270 MHz, CDCI3) 6 0.72 (s, 3H), 0.97 (d, J = 8 Hz, 3H), 1.03 (d, J - 8 Hz, 6H), 1.10 (s, 3H), 2.16 (s, 3H), 2.47 (d, J = 14 Hz, IH), 2.65 (d, J = 15 Hz, IH), 3.76 (s, 3H), 4.67 (s, IH), 4.73 (s, IH), 6.81 (t, J = 4 Hz, IH) ppm; EI-HRMS, m/z observed 498.3347 required for C3;iH4605 498.3347; EI-LRMS m/z (relative intensity) 498 (1), 483 (2), 456 (37), 382 (4), 312 (28), 287 (27), 135 (28), 123 (27), 109 (36), 95 (57), 81 (57), 69 (93), 55 (100). - 139 -APPENDIX 1 Bioassay results for phidolopin (36) and desmethylphidolopin (37) Phidolopin (36) and desmethylphidolopin (37) demonstrated significant levels of activity in the standard disk minimum inhibitory concentration bioassays performed in our laboratory. 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